Glucose-responsive insulin conjugates

ABSTRACT

Insulin conjugates comprising an insulin molecule covalently attached to at least one bi-dentate linker having two arms, each arm independently attached to a ligand comprising a saccharide and wherein the saccharide for at least one ligand of the linker is fucose are disclosed. The insulin conjugates display a pharmacokinetic (PK) and/or pharmacodynamic (PD) profile that is responsive to the systemic concentrations of a saccharide such as glucose or alpha-methylmannose even when administered to a subject in need thereof in the absence of an exogenous multivalent saccharide-binding molecule such as Con A.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.61/886,717 filed Oct. 4, 2013, and which is incoporated herein in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “23616-SEQTXT-18Feb. 2016. txt”, creation date of Feb. 18,2016, and a size of 6 KB. This sequence listing submitted via EFS-Web ispart of the specification and is herein incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to insulin conjugates comprising fucosethat display a pharmacokinetic (PK) and/or pharmacodynamic (PD) profilethat is responsive to the systemic concentrations of a saccharide suchas glucose or alpha-methylmannose even when administered to a subject inneed thereof in the absence of an exogenous multivalentsaccharide-binding molecule such as Con A. In particular, the presentinvention relates to insulin conjugates that comprise an insulinmolecule covalently attached to at least one bi-dentate linker whereineach arm of the linker is independently attached to a ligand comprisinga saccharide and wherein the saccharide for at least one ligand isfucose.

(2) Description of Related Art

The majority of “controlled-release” drug delivery systems known in theprior art (e.g., U.S. Pat. No. 4,145,410 to Sears which describes drugrelease from capsules which are enzymatically labile) are incapable ofproviding drugs to a patient at intervals and concentrations which arein direct proportion to the amount of a molecular indicator (e.g., ametabolite) present in the human body. The drugs in these prior artsystems are thus not literally “controlled,” but simply provided in aslow release format which is independent of external or internalfactors. The treatment of diabetes mellitus with injectable insulin is awell-known and studied example where uncontrolled, slow release ofinsulin is undesirable. In fact, it is apparent that the simplereplacement of the hormone is not sufficient to prevent the pathologicalsequelae associated with this disease. The development of these sequelaeis believed to reflect an inability to provide exogenous insulinproportional to varying blood glucose concentrations experienced by thepatient. To solve this problem several biological and bioengineeringapproaches to develop a more physiological insulin delivery system havebeen suggested (e.g., see U.S. Pat. No. 4,348,387 to Brownlee et al.;U.S. Pat. Nos. 5,830,506, 5,902,603, and 6,410,053 to Taylor et al. andU.S. Patent Application Publication No. 2004-0202719 to Zion et al.).

Each of these systems relies on the combination of a multivalent glucosebinding molecule (e.g., the lectin Con A) and a sugar based componentthat is reversibly bound by the multivalent glucose binding molecule.Unfortunately, Con A and many of the other readily available lectinshave the potential to stimulate lymphocyte proliferation. By binding tocarbohydrate receptors on the surfaces of particular types oflymphocytes, these so-called “mitogenic” lectins can potentially inducethe mitosis of lymphocytes and thereby cause them to proliferate. Mostmitogenic lectins including Con A are selective T-cell mitogens. A fewlectins are less selective and stimulate both T-cells and B-cells. Localor systemic in vivo exposure to mitogenic lectins can result ininflammation, cytotoxicity, macrophage digestion, and allergic reactionsincluding anaphylaxis. In addition, plant lectins are known to beparticularly immunogenic, giving rise to the production of high titersof anti-lectin specific antibodies. It will be appreciated thatmitogenic lectins cannot therefore be used in their native form for invivo methods and devices unless great care is taken to prevent theirrelease. For example, in U.S. Pat. No. 5,830,506, Taylor highlights thetoxic risks that are involved in using Con A and emphasizes theimportance and difficulty of containing Con A within a drug deliverydevice that also requires glucose and insulin molecules to diffusefreely in and out of the device. The risks and difficulties that areinvolved with these and other in vivo uses of lectins could besignificantly diminished if an alternative controlled drug deliverysystem could be provided that did not require lectins.

BRIEF SUMMARY OF THE INVENTION

The present invention provides insulin conjugates comprising fucose thatdisplay a pharmacokinetic (PK) and/or pharmacodynamic (PD) profile thatis responsive to the systemic concentrations of a saccharide such asglucose or alpha-methylmannose when administered to a subject in needthereof in the absence of an exogenous multivalent saccharide-bindingmolecule such as Con A. In general, the conjugates comprise an insulinor insulin analog molecule covalently attached to at least one branchedlinker having two arms (bi-dentate linker), each arm independentlyattached to a ligand comprising a saccharide wherein at least one ligandof the linker is fucose. In particular embodiments, the linker isnon-polymeric. In particular embodiments, a conjugate may have apolydispersity index of one and a MW of less than about 20,000 Da. Inparticular embodiments, the conjugate is long acting (i.e., exhibits aPK profile that is more sustained than soluble recombinant human insulin(RHI)).

The conjugates disclosed herein display a pharmacodynamic (PD) orpharmacokinetic (PK) profile that is sensitive to the serumconcentration of a serum saccharide when administered to a subject inneed thereof in the absence of an exogenous saccharide binding molecule.In particular aspects, the serum saccharide is glucose oralpha-methylmannose. In further aspects, the conjugate binds anendogenous saccharide binding molecule at a serum glucose concentrationof 60 or 70 mg/dL or less when administered to a subject in needthereof. The binding of the conjugate to the endogenous saccharidebinding molecule is sensitive to the serum concentration of the serumsaccharide. In a further aspect, the conjugate is capable of binding theinsulin receptor at a serum saccharide concentration great than 60, 70,80, 90, or 100 mg/dL. At serum saccharide concentration at 60 or 70mg/dL the conjugate preferentially binds the endogenous saccharidebinding molecule over the insulin receptor and as the serumconcentration of the serum saccharide increases from 60 or 70 mg/dL, thebinding of the conjugate to the endogenous saccharide binding moleculedecreases and the binding of the conjugate to the insulin receptorincreases.

Therefore, the present invention provides a conjugate comprising aninsulin or insulin analog molecule covalently attached to at least onebranched linker having a first and second arm, wherein the first arm islinked to a first ligand that includes or consists of a first saccharideand the second arm is linked to a second ligand that includes orconsists of a second saccharide, wherein the first saccharide for atleast one branched linker is fucose. Further provided is a compositioncomprising said conjugate and a pharmaceutically acceptable carrier, andoptionally one or more pharmaceutically acceptable incipiants,preservatives, zinc salt, and/or surfactants.

In particular aspects of the conjugate, the second saccharide is fucose,mannose, glucosamine, or glucose. In other aspects, the second ligandcomprises a bisaccharide, trisaccharide, tetrasaccharide, or branchedtrisaccharide. In a further aspect, the second ligand comprises abimannose, trimannose, tetramannose, or branched trimannose. Inparticular aspects, both the first saccharide and the second saccharideare fucose. In particular aspects, the first saccharide is fucose andthe second saccharide is a branched trimannose. In particular aspects,the first saccharide is fucose and the second saccharide is atrimannose. In particular aspects, the first saccharide is fucose andthe second saccharide is glucose. In particular aspects, the firstsaccharide is fucose and the second saccharide is a mannose. Inparticular aspects, the first saccharide is fucose and the secondsaccharide is a bimannose. In particular aspects, the first saccharideis fucose and the second saccharide is a trimannose. In particularaspects, the first saccharide is fucose and the second saccharide is atetramannose.

In particular aspects, the at least one branched linker is covalentlylinked to the amino acid at position A1 of the insulin or insulin analogmolecule; position B1 of the insulin or insulin analog molecule;position B29 of the insulin or insulin molecule; position B28 of theinsulin analog molecule; or position B3 of the insulin analog molecule.

In a further aspect of the conjugate, the insulin or insulin analog isfurther covalently linked to a linear or branched linker comprising aligand that includes or consists of a saccharide. In particular aspects,the saccharide is fucose, mannose, glucosamine, or glucose. In otheraspects, the ligand comprises a bisaccharide, trisaccharide,tetrasaccharide, or branched trisaccharide. In a further aspect, theligand comprises a bimannose, trimannose, tetramannose, or branchedtrimannose.

In a further aspect of the conjugate, the conjugate has the generalformula (I):

-   -   wherein:

(i) each occurrence of

represents a potential repeat within a branch of the conjugate;

(ii) each occurrence of

is independently a covalent bond, a carbon atom, a heteroatom, or anoptionally substituted group selected from the group consisting of acyl,aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic;

(iii) each occurrence of T is independently a covalent bond or abivalent, straight or branched, saturated or unsaturated, optionallysubstituted C₁₋₃₀ hydrocarbon chain wherein one or more methylene unitsof T are optionally and independently replaced by —O—, —S—, —N(R)—,—C(O)—, —C(O)O—, —OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)₂—,—N(R)SO₂—, —SO₂N(R)—, a heterocyclic group, an aryl group, or aheteroaryl group;

(iv) each occurrence of R is independently hydrogen, a suitableprotecting group, or an acyl moiety, arylalkyl moiety, aliphatic moiety,aryl moiety, heteroaryl moiety, or heteroaliphatic moiety;

(v) —B is -T-L^(B)-X, wherein each occurrence of X is independently aligand comprising a saccharide and each occurrence of L^(B) isindependently a covalent bond or a group derived from the covalentconjugation of a T with an X; and,

(vi) n is 1, 2, or 3,

with the proviso that the insulin or insulin analog is conjugated to atleast one linker in which one of the ligands X comprises a saccharide,which is fucose.

In particular aspects of the conjugate, at least one saccharide for atleast one linker is fucose and the other saccharide or saccharides arefucose, mannose, glucosamine, or glucose. In other aspects, at least onesaccharide for at least one linker is fucose and the other saccharide orsaccharides are a bisaccharide, trisaccharide, tetrasaccharide, orbranched trisaccharide. In a further aspect, at least one saccharide forat least one linker is fucose and the other saccharide or saccharidesare a bimannose, trimannose, tetramannose, or branched trimannose.Further provided is a composition comprising said conjugate and apharmaceutically acceptable carrier, and optionally one or morepharmaceutically acceptable incipiants, preservatives, zinc salt, and/orsurfactants.

In a particular aspect of the conjugate, n is 1 and the sacchride forthe first occurrence of X is fucose and the saccharide for the secondoccurrence of X is fucose, mannose, glucosamine, or glucose. In otheraspects, the sacchride for the first occurrence of X is fucose and thesaccharide for the second occurrence of X is a bisaccharide,trisaccharide, tetrasaccharide, or branched trisaccharide. In a furtheraspect, the sacchride for the first occurrence of X is fucose and thesaccharide for the second occurrence of X is a bimannose, trimannose,tetramannose, or branched trimannose. Further provided is a compositioncomprising said conjugate and a pharmaceutically acceptable carrier, andoptionally one or more pharmaceutically acceptable incipiants,preservatives, zinc salt, and/or surfactants.

In a particular aspect of the conjugate, n is 2 and the sacchride forthe first occurrence of X is fucose and the saccharide for the second,third, and fourth occurrences of X are independently fucose, mannose,glucosamine, glucose, bisaccharide, trisaccharide, tetrasaccharide, orbranched trisaccharide, bimannose, trimannose, tetramannose, or branchedtrimannose. Further provided is a composition comprising said conjugateand a pharmaceutically acceptable carrier, and optionally one or morepharmaceutically acceptable incipiants, preservatives, zinc salt, and/orsurfactants.

In a particular aspect of the conjugate, n is 3 and the sacchride forthe first occurrence of X is fucose and the saccharide for the second,third, fourth, fifth, and sixth occurrences of X are independentlyfucose, mannose, glucosamine, glucose, bisaccharide, trisaccharide,tetrasaccharide, branched trisaccharide, bimannose, trimannose,tetramannose, or branched trimannose. Further provided is a compositioncomprising said conjugate and a pharmaceutically acceptable carrier, andoptionally one or more pharmaceutically acceptable incipiants,preservatives, zinc salt, and/or surfactants.

In a further aspect of the conjugate, the conjugate comprises thegeneral formula (II):

-   -   wherein:

(i) each occurrence of

represents a potential repeat within a branch of the conjugate;

(ii) each occurrence of

is independently a covalent bond, a carbon atom, a heteroatom, or anoptionally substituted group selected from the group consisting of acyl,aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic;

(iii) each occurrence of T is independently a covalent bond or abivalent, straight or branched, saturated or unsaturated, optionallysubstituted C₁₋₃₀ hydrocarbon chain wherein one or more methylene unitsof T are optionally and independently replaced by —O—, —S—, —N(R)—,—C(O)—, —C(O)O—, —OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)₂—,—N(R)SO₂—, —SO₂N(R)—, a heterocyclic group, an aryl group, or aheteroaryl group;

(iv) each occurrence of R is independently hydrogen, a suitableprotecting group, or an acyl moiety, arylalkyl moiety, aliphatic moiety,aryl moiety, heteroaryl moiety, or heteroaliphatic moiety;

(v) —B₁ is -T-L^(B1)-X₁, wherein X₁ is a ligand comprising fucose,wherein L^(B1) is a covalent bond or a group derived from the covalentconjugation of a T with X₁;

(vi) —B₂ is -T-L^(B2)-X₂, wherein X₂ is a ligand comprising asaccharide, which may be fucose, mannose, or glucose; and L^(B2) is acovalent bond or a group derived from the covalent conjugation of a Twith an X₂; and,

(vii) n is 1, 2, or 3.

In particular aspects of the conjugate, X₂ is fucose, mannose,glucosamine, glucose, bisaccharide, trisaccharide, tetrasaccharide, orbranched trisaccharide, bimannose, trimannose, tetramannose, or branchedtrimannose. Further provided is a composition comprising said conjugateand a pharmaceutically acceptable carrier, and optionally one or morepharmaceutically acceptable incipiants, preservatives, zinc salt, and/orsurfactants.

In a further aspect of the conjugate, the bi-dentate linker has theformula

wherein each X is independently a ligand comprising a saccharide withthe proviso that at least one bi-dentate linker conjugated to theinsulin or insulin analog comprises a ligand X comprising fucose on atleast one arm of the bi-dentate linker. Further provided is acomposition comprising said conjugate having said bidentate linker and apharmaceutically acceptable carrier, and optionally one or morepharmaceutically acceptable incipiants, preservatives, zinc salt, and/orsurfactants.

In a further aspect of the conjugate, each X may independently haveformula

wherein the wavy line indicates the bond is linked to an atom comprisingthe bi-dentate linker with the proviso that at least one bi-dentatelinker conjugated to the insulin or insulin analog comprises EDF on atleast one arm of the bi-dentate linker. Further provided is acomposition comprising said conjugate and a pharmaceutically acceptablecarrier, and optionally one or more pharmaceutically acceptableincipiants, preservatives, zinc salt, and/or surfactants.

In a further aspect of the conjugate, the conjugate comprises an insulinor insulin analog molecule covalently attached to at least two branchedlinkers, each having a first and second arm, wherein the first arm islinked to a first ligand that includes a first saccharide and the secondarm is linked to a second ligand that includes a second saccharide,wherein the first saccharide is fucose. In particular aspects of theconjugate, the second saccharide is independently a fucose, mannose,glucosamine, or glucose. In other aspects, the second saccharide isindependently a bisaccharide, trisaccharide, tetrasaccharide, orbranched trisaccharide. In a further aspect, the second saccharide isindependently a bimannose, trimannose, tetramannose, or branchedtrimannose. In particular aspects, both the first saccharide and thesecond saccharide are fucose. In particular aspects, the firstsaccharide is fucose and the second saccharide is a branched trimannose.In particular aspects, the first saccharide is fucose and the secondsaccharide is a trimannose. In particular aspects, the first saccharideis fucose and the second saccharide is glucose. In particular aspects,the first saccharide is fucose and the second saccharide is a mannose.In particular aspects, the first saccharide is fucose and the secondsaccharide is a bimannose. In particular aspects, the first saccharideis fucose and the second saccharide is a trimannose. In particularaspects, the first saccharide is fucose and the second saccharide is atetramannose.

In a further aspect of the above conjugate, two amino acid positionsselected from A1, B1, B29, B28, and B3 of the insulin or insulin analogmolecule are covalently linked to the two linkers.

In a further aspect of the conjugate, the conjugate comprises an insulinor insulin analog molecule covalently attached to at least threebranched linkers, each having a first and second arm, wherein the firstarm is linked to a first ligand that includes a first saccharide and thesecond arm is linked to a second ligand that includes a secondsaccharide, wherein the first saccharide is fucose. In particularaspects of the conjugate, the second saccharide is independently afucose, mannose, glucosamine, or glucose. In other aspects, the secondsaccharide is independently a bisaccharide, trisaccharide,tetrasaccharide, or branched trisaccharide. In a further aspect, thesecond saccharide is independently a bimannose, trimannose,tetramannose, or branched trimannose. In particular aspects, both thefirst saccharide and the second saccharide are fucose. In particularaspects, the first saccharide is fucose and the second saccharide is abranched trimannose. In particular aspects, the first saccharide isfucose and the second saccharide is a trimannose. In particular aspects,the first saccharide is fucose and the second saccharide is glucose. Inparticular aspects, the first saccharide is fucose and the secondsaccharide is a mannose. In particular aspects, the first saccharide isfucose and the second saccharide is a bimannose. In particular aspects,the first saccharide is fucose and the second saccharide is atrimannose. In particular aspects, the first saccharide is fucose andthe second saccharide is a tetramannose.

In a further aspect of the above conjugate, three amino acid positionsselected from A1, B1, B29, B28, and B3 of the insulin or insulin analogmolecule are covalently linked to the three linkers.

In a further aspect of the conjugate, the conjugate comprises an insulinor insulin analog molecule covalently attached to at least two branchedlinkers, each having a first and second arm, wherein the first arm islinked to a first ligand that includes a first saccharide and the secondarm is linked to a second ligand that includes a second saccharide,wherein the first saccharide of one of the two linkers is fucose.

In particular aspects of the conjugate, the remaining first saccharideand second saccharide are independently fucose, mannose, glucosamine, orglucose. In other aspects, the remaining first saccharide and secondsaccharide are independently a bisaccharide, trisaccharide,tetrasaccharide, or branched trisaccharide. In a further aspect, theremaining first saccharide and second saccharide are independently abimannose, trimannose, tetramannose, or branched trimannose.

In particular aspects, both the first saccharide and the secondsaccharide for each of the two linkers are fucose. In particularaspects, for each of the two linkers the first saccharide is fucose andthe second saccharide is a branched trimannose. In particular aspects,for each of the two linkers the first saccharide is fucose and thesecond saccharide is a trimannose. In particular aspects, for each ofthe two linkers the first saccharide is fucose and the second saccharideis glucose. In particular aspects, for each of the two linkers the firstsaccharide for each of the two linkers is fucose and the secondsaccharide is a mannose. In particular aspects, for each of the twolinkers the first saccharide is fucose and the second saccharide is abimannose. In particular aspects, for each of the two linkers the firstsaccharide is fucose and the second saccharide is a trimannose. Inparticular aspects, for each of the two linkers the first saccharide isfucose and the second saccharide is a tetramannose.

In particular aspects, both the first saccharide and the secondsaccharide for one of the two linkers are fucose and for the secondlinker the first saccharide and second saccharide are independentlyfucose, mannose, glucosamine, glucose bisaccharide, trisaccharide,tetrasaccharide, or branched trisaccharide bimannose, trimannose,tetramannose, or branched trimannose. In particular aspects, for one ofthe two linkers the first saccharide is fucose and the second saccharideis a branched trimannose and for the second linker the first saccharideand second saccharide are independently fucose, mannose, glucosamine,glucose bisaccharide, trisaccharide, tetrasaccharide, or branchedtrisaccharide bimannose, trimannose, tetramannose, or branchedtrimannose. In particular aspects, for one of the two linkers the firstsaccharide is fucose and the second saccharide is a trimannose and forthe second linker the first saccharide and second saccharide areindependently fucose, mannose, glucosamine, glucose bisaccharide,trisaccharide, tetrasaccharide, or branched trisaccharide bimannose,trimannose, tetramannose, or branched trimannose. In particular aspects,for one of the two linkers the first saccharide is fucose and the secondsaccharide is glucose and for the second linker the first saccharide andsecond saccharide are independently fucose, mannose, glucosamine,glucose bisaccharide, trisaccharide, tetrasaccharide, or branchedtrisaccharide bimannose, trimannose, tetramannose, or branchedtrimannose. In particular aspects, for one of the two linkers the firstsaccharide for each of the two linkers is fucose and the secondsaccharide is a mannose and for the second linker the first saccharideand second saccharide are independently fucose, mannose, glucosamine,glucose bisaccharide, trisaccharide, tetrasaccharide, or branchedtrisaccharide bimannose, trimannose, tetramannose, or branchedtrimannose. In particular aspects, for one of the two linkers the firstsaccharide is fucose and the second saccharide is a bimannose and forthe second linker the first saccharide and second saccharide areindependently fucose, mannose, glucosamine, glucose bisaccharide,trisaccharide, tetrasaccharide, or branched trisaccharide bimannose,trimannose, tetramannose, or branched trimannose. In particular aspects,for one of the two linkers the first saccharide is fucose and the secondsaccharide is a trimannose and for the second linker the firstsaccharide and second saccharide are independently fucose, mannose,glucosamine, glucose bisaccharide, trisaccharide, tetrasaccharide, orbranched trisaccharide bimannose, trimannose, tetramannose, or branchedtrimannose. In particular aspects, for one of the two linkers the firstsaccharide is fucose and for the second linker the second saccharide isa tetramannose and the first saccharide and second saccharide areindependently fucose, mannose, glucosamine, glucose bisaccharide,trisaccharide, tetrasaccharide, or branched trisaccharide bimannose,trimannose, tetramannose, or branched trimannose.

In a further aspect of the above conjugate, two amino acid positionsselected from A1, B1, B29, B28, and B3 of the insulin or insulin analogmolecule are covalently linked to the two linkers.

In a further aspect of the conjugate, the conjugate comprises an insulinor insulin analog molecule covalently attached to at least threebranched linkers, each having a first and second arm, wherein the firstarm is linked to a first ligand that includes a first saccharide and thesecond arm is linked to a second ligand that includes a secondsaccharide, wherein the first saccharide of at least one of the threelinkers is fucose and the remaining first saccharides and secondsaccharide are independently fucose, mannose, glucosamine, or glucose.In other aspects, the remaining first saccharides and second saccharideare independently a bisaccharide, trisaccharide, tetrasaccharide, orbranched trisaccharide. In a further aspect, the remaining firstsaccharides and second saccharide are a bimannose, trimannose,tetramannose, or branched trimannose.

In a further aspect of the above conjugate, three amino acid positionsselected from A1, B1, B29, B28, and B3 of the insulin or insulin analogmolecule are covalently linked to the three linkers.

Further provided is conjugate or a composition comprising a conjugatehaving the formula as set forth for IOC-1, IOC-2, IOC-3, IOC-4, IOC-5,IOC-6, IOC-7, IOC-8, IOC-9, IOC-10, IOC-11, IOC-12, IOC-13, IOC-14,IOC-15, IOC-16, IOC-17, IOC-18, IOC-19, IOC-20, IOC-21, IOC-22, IOC-23,IOC-24, IOC-25, IOC-26, IOC-27, IOC-28, IOC-29, IOC-30, IOC-31, IOC-32,IOC-33, IOC-34, IOC-35, IOC-36, IOC-37, IOC-38, IOC-39, IOC-41, IOC-42,IOC-43, IOC-44, IOC-45, IOC-46, IOC-47, IOC-49, IOC-50, IOC-51, IOC-52,IOC-53, IOC-54, IOC-55, IOC-56, IOC-57, IOC-58, IOC-59, IOC-60, IOC-61,IOC-62, IOC-63, IOC-64, IOC-65, IOC-66, IOC-67, IOC-68, IOC-69, IOC-70,IOC-71, IOC-72, IOC-73, IOC-74, IOC-75, IOC-76, IOC-77, IOC-78, IOC-79,IOC-80, IOC-81, IOC-82, IOC-83, IOC-84, IOC-85, IOC-86, IOC-87, IOC-88,IOC-89, IOC-90, IOC-91, IOC-92, IOC-93, IOC-94, IOC-95, IOC-96, IOC-97,IOC-98, IOC-99, or IOC-100.

Further provided is conjugate or a composition comprising a conjugatehaving the formula as set forth for IOC-101, IOC-102, IOC-103, IOC-104,IOC-105, IOC-106, IOC-107, IOC-108, IOC-109, IOC-110, IOC-111, IOC-112,IOC-113, IOC-114, IOC-115, IOC-116, IOC-117, IOC-118, IOC-119, IOC-120,IOC-121, IOC-122, IOC-123, IOC-124, IOC-125, IOC-126, IOC-127, IOC-128,IOC-129, IOC-130, IOC-131, IOC-132, IOC-133, IOC-134, IOC-135, IOC-136,IOC-137, IOC-138, IOC-139, IOC-140, IOC-141, IOC-142, IOC-143, IOC-144,IOC-145, IOC-146, IOC-147, IOC-149, IOC-150, IOC-151, IOC-152, IOC-153,IOC-154, IOC-155, IOC-156, IOC-157, IOC-158, IOC-159, IOC-160, IOC-161,IOC-162, IOC-163, IOC-164, IOC-165, IOC-166, IOC-167, IOC-168, IOC-169,IOC-170, IOC-171, IOC-172, IOC-173, IOC-174, IOC-175, IOC-176, IOC-177,IOC-178, IOC-179, IOC-180, IOC-181, IOC-182, IOC-183, IOC-184, IOC-185,IOC-186, IOC-187, IOC-188, IOC-189, IOC-190, IOC-191, or IOC-192.

Further provided is conjugate or a composition comprising a conjugatehaving the formula as set forth for IOC-193, IOC-194, IOC-195, IOC-196,IOC-197, IOC-198, IOC-199, IOC-200, IOC-201, IOC-202, IOC-203, IOC-204,IOC-205, IOC-206, IOC-207, IOC-208, IOC-210, IOC-211, IOC-212, IOC-213,IOC-214, IOC-215, IOC-216, IOC-217, IOC-218, IOC-219, IOC-220, IOC-221,IOC-222, IOC-223, IOC-224, IOC-225, IOC-226, IOC-227, IOC-228, IOC-229,IOC-230, IOC-231, IOC-232, IOC-233, IOC-234, IOC-235, IOC-236, IOC-237,IOC-238, IOC-239, IOC-240, IOC-241, IOC-242, IOC-243, IOC-244, IOC-245,IOC-246, IOC-247, IOC-248, IOC-249, IOC-250, IOC-251, IOC-252, IOC-253,IOC-254, IOC-255, IOC-256, IOC-257, IOC-258, IOC-259, IOC-260, IOC-261,IOC-262, IOC-263, IOC-264, IOC-265, IOC-266, IOC-267, IOC-268, IOC-269,IOC-270, IOC-271, or IOC-272.

The above conjugates may further be provided in a pharmaceutical formulacomprising a pharmaceutically acceptable carrier and optionally one ormore pharmaceutically acceptable excipients, preservatives, and/orsurfactants. In further aspects the conjugates may further include zincand/or a protamine. The conjugates may be provided as crystalline form.

Thus, the present invention further provides a composition comprisingone or more of the conjugates as generically or specifically disclosedherein and a pharmaceutically acceptable carrier, and optionally one ormore pharmaceutically acceptable incipiants, preservatives, zinc salt,and/or surfactants. Further provided is a composition comprising one ormore of the conjugates as generically or specifically disclosed hereinin a crystalline form and a pharmaceutically acceptable carrier, andoptionally one or more pharmaceutically acceptable incipiants,preservatives, zinc salt, protamine, and/or surfactants.

In further aspects of the conjugates, the insulin molecule is amammalian insulin, which in particular embodiments, may be a humaninsulin, bovine insulin, dog insulin, cat insulin, goat insulin, horseinsulin, pig insulin or an analog thereof. In particular embodiments,the insulin analog is insulin lispro or insulin glulisine. In furtherembodiments, the insulin or insulin analog is modified to comprise apolyethylene glycol or fatty acid covalently linked to an amino acid ofthe insulin or insulin analog. In particular embodiments, the insulinanalog is insulin lispro, insulin glargine, insulin aspart, insulindetemir, or insulin glulisine.

The present invention further provides methods for treating diabetescomprising administering the conjugates disclosed herein or apharmaceutical formulation comprising the conjugates disclosed herein toan subject who is diabetic. The present invention further provides forthe use of the conjugates disclosed herein for the manufacture of amedicament for the treatment of diabetes.

The present invention further provides a composition or pharmaceuticalcomposition comprising: an insulin or insulin analog molecule covalentlyattached to at least one branched linker having a first arm and secondarm, wherein the first arm is linked to a first ligand that includes afirst saccharide and the second arm is linked to a second ligand thatincludes a second saccharide and wherein the first saccharide is fucose,to provide a conjugate, and a pharmaceutically acceptable carrier.

In general, the second saccharide is a fucose, mannose, glucosamine,glucose, bisaccharide, trisaccharide, tetrasaccharide, branchedtrisaccharide, bimannose, trimannose, tetramannose, or branchedtrimannose.

In particular aspects, the branched linker is covalently linked to theamino acid at position A1 of the insulin or insulin analog molecule;position B1 of the insulin or insulin analog molecule; position B29 ofthe insulin or insulin molecule; position B28 of the insulin analogmolecule; or position B3 of the insulin analog molecule.

In a further embodiment, the insulin or insulin analog is covalentlyattached to a second branched linker having a first arm and second arm,wherein the first arm is linked to a third ligand that includes a thirdsaccharide and the second arm is linked to a fourth ligand that includesa fourth saccharide. In particular aspects, the second branched linkeris covalently linked to the amino acid at position A1 of the insulin orinsulin analog molecule; position B1 of the insulin or insulin analogmolecule; position B29 of the insulin or insulin molecule; position B28of the insulin analog molecule; or position B3 of the insulin analogmolecule and which is not occupied by the first branched linker.

In a further embodiment, the insulin or insulin analog is covalentlyattached to a third branched linker having a first arm and second arm,wherein the first arm is linked to a fifth ligand that includes a fifthsaccharide and the second arm is linked to a sixth ligand that includesa sixth saccharide. In particular aspects, the third branched linker iscovalently linked to the amino acid at position A1 of the insulin orinsulin analog molecule; position B1 of the insulin or insulin analogmolecule; position B29 of the insulin or insulin molecule; position B28of the insulin analog molecule; or position B3 of the insulin analogmolecule and which is not occupied by the first branched linker and thesecond branched linker.

In any embodiment of the above conjugate, the third, fourth, fifth, andsixth saccharides are each independently a fucose, mannose, glucosamine,glucose, bisaccharide, trisaccharide, tetrasaccharide, branchedtrisaccharide, bimannose, trimannose, tetramannose, or branchedtrimannose.

In a further aspect, the insulin or insulin analog molecule is furthercovalently linked to a linear linker comprising a ligand that includes asaccharide and the saccharide may be a fucose, mannose, glucosamine,glucose, bisaccharide, trisaccharide, tetrasaccharide, branchedtrisaccharide, bimannose, trimannose, tetramannose, or branchedtrimannose.

In particular aspects, the insulin analog molecule is insulin lispro,insulin glargine, insulin aspart, insulin detemir, or insulin glulisine.

In any one of the above aspects or embodiments, the conjugate displays apharmacodynamic (PD) or pharmacokinetic (PK) profile that is sensitiveto the serum concentration of a serum saccharide when administered to asubject in need thereof in the absence of an exogenous saccharidebinding molecule. In particular aspects, the serum saccharide is glucoseor alpha-methylmannose. In particular aspects, the conjugate binds anendogenous saccharide binding molecule at a serum glucose concentrationof 60 mg/dL or less when administered to a subject in need thereof. Theendogenous saccharide binding molecule may be the human mannose receptor1.

In particular aspects of the composition, the conjugate has the generalformula (I):

-   -   wherein:    -   (i) each occurrence of

represents a potential repeat within a branch of the conjugate;

-   -   (ii) each occurrence of        is independently a covalent bond, a carbon atom, a heteroatom,        or an optionally substituted group selected from the group        consisting of acyl, aliphatic, heteroaliphatic, aryl,        heteroaryl, and heterocyclic;    -   (iii) each occurrence of T is independently a covalent bond or a        bivalent, straight or branched, saturated or unsaturated,        optionally substituted C₁₋₃₀ hydrocarbon chain wherein one or        more methylene units of T are optionally and independently        replaced by —O—, —S—, —N(R)—, —C(O)—, —C(O)O—, —OC(O)—,        —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)₂—, —N(R)SO₂—, —SO₂N(R)—, a        heterocyclic group, an aryl group, or a heteroaryl group;    -   (iv) each occurrence of R is independently hydrogen, a suitable        protecting group, or an acyl moiety, arylalkyl moiety, aliphatic        moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic        moiety;    -   (v) —B is -T-L^(B)-X, wherein each occurrence of X is        independently a ligand comprising a saccharide and each        occurrence of L^(B) is independently a covalent bond or a group        derived from the covalent conjugation of a T with an X; and,    -   (vi) n is 1, 2, or 3,    -   with the proviso that at least one X is fucose.

In particular aspects of the composition, the conjugate comprises thegeneral formula (II):

-   -   wherein:    -   (i) each occurrence of

represents a potential repeat within a branch of the conjugate;

-   -   (ii) each occurrence of        is independently a covalent bond, a carbon atom, a heteroatom,        or an optionally substituted group selected from the group        consisting of acyl, aliphatic, heteroaliphatic, aryl,        heteroaryl, and heterocyclic;    -   (iii) each occurrence of T is independently a covalent bond or a        bivalent, straight or branched, saturated or unsaturated,        optionally substituted C₁₋₃₀ hydrocarbon chain wherein one or        more methylene units of T are optionally and independently        replaced by —O—, —S—, —N(R)—, —C(O)—, —C(O)O—, —OC(O)—,        —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)₂—, —N(R)SO₂—, —SO₂N(R)—, a        heterocyclic group, an aryl group, or a heteroaryl group;    -   (iv) each occurrence of R is independently hydrogen, a suitable        protecting group, or an acyl moiety, arylalkyl moiety, aliphatic        moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic        moiety;    -   (v) —B₁ is -T-L^(B1)-Fucose, wherein L^(B1) is a covalent bond        or a group derived from the covalent conjugation of a T with an        X;    -   (vi) —B₂ is -T-L^(B2)-X, wherein X is a ligand comprising a        saccharide, which may be fucose, mannose, or glucose; and L^(B2)        is a covalent bond or a group derived from the covalent        conjugation of a T with an X; and,    -   (vii) n is 1, 2, or 3.

In particular aspects of the composition, the bi-dentate linker hasformula A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U,V, W, X, Y, Z, AA, AB, AC, AD, AE, AF, AG, AH, AI, AJ, or AK as shownsupra wherein each X is independently a ligand comprising a saccharidewith the proviso that at least one bi-dentate linker conjugated to theinsulin or insulin analog comprises a fucose on at least one arm of thebi-dentate linker. In particular aspects, each X may independently haveformular EG, EM, EBM, EGA, EF, EFβ, EBM, ETM, EDG, EDF, or EDM as shownsupra.

Further provided is a composition comprising a conjugate having theformula as set forth for IOC-1, IOC-2, IOC-3, IOC-4, IOC-5, IOC-6,IOC-7, IOC-8, IOC-9, IOC-10, IOC-11, IOC-12, IOC-13, IOC-14, IOC-15,IOC-16, IOC-17, IOC-18, IOC-19, IOC-20, IOC-21, IOC-22, IOC-23, IOC-24,IOC-25, IOC-26, IOC-27, IOC-28, IOC-29, IOC-30, IOC-31, IOC-32, IOC-33,IOC-34, IOC-35, IOC-36, IOC-37, IOC-38, IOC-39, IOC-41, IOC-42, IOC-43,IOC-44, IOC-45, IOC-46, IOC-47, IOC-49, IOC-50, IOC-51, IOC-52, IOC-53,IOC-54, IOC-55, IOC-56, IOC-57, IOC-58, IOC-59, IOC-60, IOC-61, IOC-62,IOC-63, IOC-64, IOC-65, IOC-66, IOC-67, IOC-68, IOC-69, IOC-70, IOC-71,IOC-72, IOC-73, IOC-74, IOC-75, IOC-76, IOC-77, IOC-78, IOC-79, IOC-80,IOC-81, IOC-82, IOC-83, IOC-84, IOC-85, IOC-86, IOC-87, IOC-88, IOC-89,IOC-90, IOC-91, IOC-92, IOC-93, IOC-94, IOC-95, IOC-96, IOC-97, IOC-98,IOC-99, or IOC-100, and a pharmaceutically acceptable carrier; acomposition comprising a conjugate having the formula as set forth forIOC-101, IOC-102, IOC-103, IOC-104, IOC-105, IOC-106, IOC-107, IOC-108,IOC-109, IOC-110, IOC-111, IOC-112, IOC-113, IOC-114, IOC-115, IOC-116,IOC-117, IOC-118, IOC-119, IOC-120, IOC-121, IOC-122, IOC-123, IOC-124,IOC-125, IOC-126, IOC-127, IOC-128, IOC-129, IOC-130, IOC-131, IOC-132,IOC-133, IOC-134, IOC-135, IOC-136, IOC-137, IOC-138, IOC-139, IOC-140,IOC-141, IOC-142, IOC-143, IOC-144, IOC-145, IOC-146, IOC-147, IOC-149,IOC-150, IOC-151, IOC-152, IOC-153, IOC-154, IOC-155, IOC-156, IOC-157,IOC-158, IOC-159, IOC-160, IOC-161, IOC-162, IOC-163, IOC-164, IOC-165,IOC-166, IOC-167, IOC-168, IOC-169, IOC-170, IOC-171, IOC-172, IOC-173,IOC-174, IOC-175, IOC-176, IOC-177, IOC-178, IOC-179, IOC-180, IOC-181,IOC-182, IOC-183, IOC-184, IOC-185, IOC-186, IOC-187, IOC-188, IOC-189,IOC-190, IOC-191, or IOC-192, and a pharmaceutically acceptable carrier;and a composition comprising a conjugate having the formula as set forthfor IOC-193, IOC-194, IOC-195, IOC-196, IOC-197, IOC-198, IOC-199,IOC-200, IOC-201, IOC-202, IOC-203, IOC-204, IOC-205, IOC-206, IOC-207,IOC-208, IOC-210, IOC-211, IOC-212, IOC-213, IOC-214, IOC-215, IOC-216,IOC-217, IOC-218, IOC-219, IOC-220, IOC-221, IOC-222, IOC-223, IOC-224,IOC-225, IOC-226, IOC-227, IOC-228, IOC-229, IOC-230, IOC-231, IOC-232,IOC-233, IOC-234, IOC-235, IOC-236, IOC-237, IOC-238, IOC-239, IOC-240,IOC-241, IOC-242, IOC-243, IOC-244, IOC-245, IOC-246, IOC-247, IOC-248,IOC-249, IOC-250, IOC-251, IOC-252, IOC-253, IOC-254, IOC-255, IOC-256,IOC-257, IOC-258, IOC-259, IOC-260, IOC-261, IOC-262, IOC-263, IOC-264,IOC-265, IOC-266, IOC-267, IOC-268, IOC-269, IOC-270, IOC-271, orIOC-272, and a pharmaceutically acceptable carrier.

The present invention further provides for the use of the compositionsdisclosed herein for the treatment of diabetes. In particular aspects,the diabetes is type I diabetes, type II diabetes, or gestationaldiabetes.

The present invention further provides a method for treating a subjectwho has diabetes comprising: administering to the subject an amount of acomposition for treating the diaabetes, wherein the compositioncomprises an insulin or insulin analog molecule covalently attached toat least one branched linker having a first arm and second arm, whereinthe first arm is linked to a first ligand that includes a firstsaccharide and the second arm is linked to a second ligand that includesa second saccharide and wherein the first saccharide is fucose, toprovide a conjugate, and a pharmaceutically acceptable carrier to treatthe diabetes; wherein said administering treats the diabetes. Inparticular aspects, the amount of composition administered is aneffective amount or therapeutically effective amount.

In general, the second saccharide is a fucose, mannose, glucosamine,glucose, bisaccharide, trisaccharide, tetrasaccharide, branchedtrisaccharide, bimannose, trimannose, tetramannose, or branchedtrimannose.

In particular aspects, the branched linker is covalently linked to theamino acid at position A1 of the insulin or insulin analog molecule;position B1 of the insulin or insulin analog molecule; position B29 ofthe insulin or insulin molecule; position B28 of the insulin analogmolecule; or position B3 of the insulin analog molecule.

In a further embodiment, the insulin or insulin analog is covalentlyattached to a second branched linker having a first arm and second arm,wherein the first arm is linked to a third ligand that includes a thirdsaccharide and the second arm is linked to a fourth ligand that includesa fourth saccharide. In particular aspects, the second branched linkeris covalently linked to the amino acid at position A1 of the insulin orinsulin analog molecule; position B1 of the insulin or insulin analogmolecule; position B29 of the insulin or insulin molecule; position B28of the insulin analog molecule; or position B3 of the insulin analogmolecule and which is not occupied by the first branched linker.

In a further embodiment, the insulin or insulin analog is covalentlyattached to a third branched linker having a first arm and second arm,wherein the first arm is linked to a fifth ligand that includes a fifthsaccharide and the second arm is linked to a sixth ligand that includesa sixth saccharide. In particular aspects, the third branched linker iscovalently linked to the amino acid at position A1 of the insulin orinsulin analog molecule; position B1 of the insulin or insulin analogmolecule; position B29 of the insulin or insulin molecule; position B28of the insulin analog molecule; or position B3 of the insulin analogmolecule and which is not occupied by the first branched linker and thesecond branched linker.

In any embodiment of the above conjugate, the third, fourth, fifth, andsixth saccharides are each independently a fucose, mannose, glucosamine,glucose, bisaccharide, trisaccharide, tetrasaccharide, branchedtrisaccharide, bimannose, trimannose, tetramannose, or branchedtrimannose.

In a further aspect, the insulin or insulin analog molecule is furthercovalently linked to a linear linker comprising a ligand that includes asaccharide and the saccharide may be a fucose, mannose, glucosamine,glucose, bisaccharide, trisaccharide, tetrasaccharide, branchedtrisaccharide, bimannose, trimannose, tetramannose, or branchedtrimannose.

In particular aspects, the insulin analog molecule is insulin lispro,insulin glargine, insulin aspart, insulin detemir, or insulin glulisine.

In any one of the above aspects or embodiments, the conjugate displays apharmacodynamic (PD) or pharmacokinetic (PK) profile that is sensitiveto the serum concentration of a serum saccharide when administered to asubject in need thereof in the absence of an exogenous saccharidebinding molecule. In particular aspects, the serum saccharide is glucoseor alpha-methylmannose. In particular aspects, the conjugate binds anendogenous saccharide binding molecule at a serum glucose concentrationof 60 mg/dL or less when administered to a subject in need thereof. Theendogenous saccharide binding molecule may be the human mannose receptor1.

In particular aspects of the composition, the conjugate has the generalformula (I):

-   -   wherein:    -   (i) each occurrence of

represents a potential repeat within a branch of the conjugate;

-   -   (ii) each occurrence of        is independently a covalent bond, a carbon atom, a heteroatom,        or an optionally substituted group selected from the group        consisting of acyl, aliphatic, heteroaliphatic, aryl,        heteroaryl, and heterocyclic;    -   (iii) each occurrence of T is independently a covalent bond or a        bivalent, straight or branched, saturated or unsaturated,        optionally substituted C₁₋₃₀ hydrocarbon chain wherein one or        more methylene units of T are optionally and independently        replaced by —O—, —S—, —N(R)—, —C(O)—, —C(O)O—, —OC(O)—,        —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)₂—, —N(R)SO₂—, —SO₂N(R)—, a        heterocyclic group, an aryl group, or a heteroaryl group;    -   (iv) each occurrence of R is independently hydrogen, a suitable        protecting group, or an acyl moiety, arylalkyl moiety, aliphatic        moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic        moiety;    -   (v) —B is -T-L^(B)-X, wherein each occurrence of X is        independently a ligand comprising a saccharide and each        occurrence of L^(B) is independently a covalent bond or a group        derived from the covalent conjugation of a T with an X; and,    -   (vi) n is 1, 2, or 3,    -   with the proviso that at least one X is fucose.

In particular aspects of the composition, the conjugate comprises thegeneral formula (II):

-   -   wherein:    -   (i) each occurrence of

represents a potential repeat within a branch of the conjugate;

-   -   (ii) each occurrence of        is independently a covalent bond, a carbon atom, a heteroatom,        or an optionally substituted group selected from the group        consisting of acyl, aliphatic, heteroaliphatic, aryl,        heteroaryl, and heterocyclic;    -   (iii) each occurrence of T is independently a covalent bond or a        bivalent, straight or branched, saturated or unsaturated,        optionally substituted C₁₋₃₀ hydrocarbon chain wherein one or        more methylene units of T are optionally and independently        replaced by —O—, —S—, —N(R)—, —C(O)—, —C(O)O—, —OC(O)—,        —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)₂—, —N(R)SO₂—, —SO₂N(R)—, a        heterocyclic group, an aryl group, or a heteroaryl group;    -   (iv) each occurrence of R is independently hydrogen, a suitable        protecting group, or an acyl moiety, arylalkyl moiety, aliphatic        moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic        moiety;    -   (v) —B₁ is -T-L^(B1)-Fucose, wherein L^(B1) is a covalent bond        or a group derived from the covalent conjugation of a T with an        X;    -   (vi) —B₂ is -T-L^(B2)-X, wherein X is a ligand comprising a        saccharide, which may be fucose, mannose, or glucose; and L^(B2)        is a covalent bond or a group derived from the covalent        conjugation of a T with an X; and,    -   (vii) n is 1, 2, or 3.

In particular aspects of the composition, the bi-dentate linker hasformula A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U,V, W, X, Y, Z, AA, AB, AC, AD, AE, AF, AG, AH, AI, AJ, or AK as shownsupra wherein each X is independently a ligand comprising a saccharidewith the proviso that at least one bi-dentate linker conjugated to theinsulin or insulin analog comprises a fucose on at least one arm of thebi-dentate linker. In particular aspects, each X may independently haveformular EG, EM, EBM, EGA, EF, EFβ, EBM, ETM, EDG, EDF, or EDM as shownsupra.

The present invention further provides a method for treating a subjectwho has diabetes, comprising: administering to the subject a compositioncomprising a conjugate having the formula as set forth for IOC-1, IOC-2,IOC-3, IOC-4, IOC-5, IOC-6, IOC-7, IOC-8, IOC-9, IOC-10, IOC-11, IOC-12,IOC-13, IOC-14, IOC-15, IOC-16, IOC-17, IOC-18, IOC-19, IOC-20, IOC-21,IOC-22, IOC-23, IOC-24, IOC-25, IOC-26, IOC-27, IOC-28, IOC-29, IOC-30,IOC-31, IOC-32, IOC-33, IOC-34, IOC-35, IOC-36, IOC-37, IOC-38, IOC-39,IOC-41, IOC-42, IOC-43, IOC-44, IOC-45, IOC-46, IOC-47, IOC-49, IOC-50,IOC-51, IOC-52, IOC-53, IOC-54, IOC-55, IOC-56, IOC-57, IOC-58, IOC-59,IOC-60, IOC-61, IOC-62, IOC-63, IOC-64, IOC-65, IOC-66, IOC-67, IOC-68,IOC-69, IOC-70, IOC-71, IOC-72, IOC-73, IOC-74, IOC-75, IOC-76, IOC-77,IOC-78, IOC-79, IOC-80, IOC-81, IOC-82, IOC-83, IOC-84, IOC-85, IOC-86,IOC-87, IOC-88, IOC-89, IOC-90, IOC-91, IOC-92, IOC-93, IOC-94, IOC-95,IOC-96, IOC-97, IOC-98, IOC-99, or IOC-100 and a pharmaceuticallyacceptable carrier to treat the diabetes in the subject; a method fortreating a subject who has diabetes, comprising: administering to thesubject a composition comprising a conjugate having the formula as setforth for IOC-101, IOC-102, IOC-103, IOC-104, IOC-105, IOC-106, IOC-107,IOC-108, IOC-109, IOC-110, IOC-111, IOC-112, IOC-113, IOC-114, IOC-115,IOC-116, IOC-117, IOC-118, IOC-119, IOC-120, IOC-121, IOC-122, IOC-123,IOC-124, IOC-125, IOC-126, IOC-127, IOC-128, IOC-129, IOC-130, IOC-131,IOC-132, IOC-133, IOC-134, IOC-135, IOC-136, IOC-137, IOC-138, IOC-139,IOC-140, IOC-141, IOC-142, IOC-143, IOC-144, IOC-145, IOC-146, IOC-147,IOC-149, IOC-150, IOC-151, IOC-152, IOC-153, IOC-154, IOC-155, IOC-156,IOC-157, IOC-158, IOC-159, IOC-160, IOC-161, IOC-162, IOC-163, IOC-164,IOC-165, IOC-166, IOC-167, IOC-168, IOC-169, IOC-170, IOC-171, IOC-172,IOC-173, IOC-174, IOC-175, IOC-176, IOC-177, IOC-178, IOC-179, IOC-180,IOC-181, IOC-182, IOC-183, IOC-184, IOC-185, IOC-186, IOC-187, IOC-188,IOC-189, IOC-190, IOC-191, or IOC-192 and a pharmaceutically acceptablecarrier to treat the diabetes in the subject; and a method for treatinga subject who has diabetes, comprising: administering to the subject acomposition comprising a conjugate having the formula as set forth forIOC-193, IOC-194, IOC-195, IOC-196, IOC-197, IOC-198, IOC-199, IOC-200,IOC-201, IOC-202, IOC-203, IOC-204, IOC-205, IOC-206, IOC-207, IOC-208,IOC-210, IOC-211, IOC-212, IOC-213, IOC-214, IOC-215, IOC-216, IOC-217,IOC-218, IOC-219, IOC-220, IOC-221, IOC-222, IOC-223, IOC-224, IOC-225,IOC-226, IOC-227, IOC-228, IOC-229, IOC-230, IOC-231, IOC-232, IOC-233,IOC-234, IOC-235, IOC-236, IOC-237, IOC-238, IOC-239, IOC-240, IOC-241,IOC-242, IOC-243, IOC-244, IOC-245, IOC-246, IOC-247, IOC-248, IOC-249,IOC-250, IOC-251, IOC-252, IOC-253, IOC-254, IOC-255, IOC-256, IOC-257,IOC-258, IOC-259, IOC-260, IOC-261, IOC-262, IOC-263, IOC-264, IOC-265,IOC-266, IOC-267, IOC-268, IOC-269, IOC-270, IOC-271, or IOC-272, and apharmaceutically acceptable carrier to treat the diabetes in thesubject.

In anyone of the aforementioned aspects or embodiments of the method,the diabetes is type I diabetes, type II diabetes, or gestationaldiabetes.

The present invention further provides a composition comprising aninsulin and insulin analog conjugate wherein the conjugate comprises atleast one fucose molecule and the conjugate is characterized as having aratio of EC₅₀ or IP as determined by a functional insulin receptorphosphorylation assay to the IC₅₀ or IP as determined by a competitionbinding assay at the macrophage mannose receptor that is about 0.5:1 toabout 1:100; about 1:1 to about 1:50; about 1:1 to about 1:20; or about1:1 to about 1:10; and a pharmaceutically acceptable carrier.

In particular aspects, wherein the conjugate comprises an insulin orinsulin analog molecule covalently attached to at least one branchedlinker having a first arm and second arm, wherein the first arm islinked to a first ligand that includes a first saccharide and the secondarm is linked to a second ligand that includes a second saccharide andwherein the first saccharide is fucose. In further aspects, the secondsaccharide is a fucose, mannose, glucosamine, glucose, bisaccharide,trisaccharide, tetrasaccharide, branched trisaccharide, bimannose,trimannose, tetramannose, or branched trimannose.

In particular embodiments, the conjugate may be a conjugate as disclosedherein.

The present invention further provides a method for treating a subjectwho has diabetes, comprising administering to the subject a compositioncomprising a conjugate comprising fucose and characterized as having aratio of EC₅₀ or IP as determined by a functional insulin receptorphosphorylation assay to the IC₅₀ or IP as determined by a competitionbinding assay at the macrophage mannose receptor that is about 0.5:1 toabout 1:100; about 1:1 to about 1:50; about 1:1 to about 1:20; or about1:1 to about 1:10; and a pharmaceutically acceptable carrier to treatthe diabetes.

In particular embodiments, the conjugate comprises an insulin or insulinanalog molecule covalently attached to at least one branched linkerhaving a first arm and second arm, wherein the first arm is linked to afirst ligand that includes a first saccharide and the second arm islinked to a second ligand that includes a second saccharide and whereinthe first saccharide is fucose. In further aspects, the secondsaccharide is a fucose, mannose, glucosamine, glucose, bisaccharide,trisaccharide, tetrasaccharide, branched trisaccharide, bimannose,trimannose, tetramannose, or branched trimannose.

In particular embodiments, the conjugate may be a conjugate as disclosedherein.

In particular aspects. the diabetes is type I diabetes, type IIdiabetes, or gestational diabetes.

DEFINITIONS

Definitions of specific functional groups, chemical terms, and generalterms used throughout the specification are described in more detailbelow. For purposes of this invention, the chemical elements areidentified in accordance with the Periodic Table of the Elements, CASversion, Handbook of Chemistry and Physics, 75^(th) Ed., inside cover,and specific functional groups are generally defined as describedtherein. Additionally, general principles of organic chemistry, as wellas specific functional moieties and reactivity, are described in OrganicChemistry, Thomas Sorrell, University Science Books, Sausalito, 1999;Smith and March March's Advanced Organic Chemistry, 5^(th) Edition, JohnWiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; Carruthers, SomeModern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Acyl—As used herein, the term “acyl,” refers to a group having thegeneral formula —C(═O)R^(X1), —C(═O)OR^(X1), —C(═O)—O—C(═O)R^(X1),—C(═O)SR^(X1), —C(═O)N(R^(X1))₂, —C(═S)R^(X1), —C(═S)N(R^(X1))₂, and—C(═S)S(R^(X1)), —C(═NR^(X1))R^(X1), —C(═NR^(X1))OR^(X1),—C(═NR^(X1))SR^(X1), and —C(═NR^(X1))N(R^(X1))₂, wherein R^(X1) ishydrogen; halogen; substituted or unsubstituted hydroxyl; substituted orunsubstituted thiol; substituted or unsubstituted amino; substituted orunsubstituted acyl; cyclic or acyclic, substituted or unsubstituted,branched or unbranched aliphatic; cyclic or acyclic, substituted orunsubstituted, branched or unbranched heteroaliphatic; cyclic oracyclic, substituted or unsubstituted, branched or unbranched alkyl;cyclic or acyclic, substituted or unsubstituted, branched or unbranchedalkenyl; substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl,aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy,heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy,heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- ordi-aliphaticamino, mono- or di-heteroaliphaticamino, mono- ordi-alkylamino, mono- or di-heteroalkylamino, mono- or di-arylamino, ormono- or di-heteroarylamino; or two R^(X1) groups taken together form a5- to 6-membered heterocyclic ring. Exemplary acyl groups includealdehydes (—CHO), carboxylic acids (—CO₂H), ketones, acyl halides,esters, amides, imines, carbonates, carbamates, and ureas. Acylsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido,nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl,arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy,aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy,alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy,and the like, each of which may or may not be further substituted).

Aliphatic—As used herein, the term “aliphatic” or “aliphatic group”denotes an optionally substituted hydrocarbon moiety that may bestraight-chain (i.e., unbranched), branched, or cyclic (“carbocyclic”)and may be completely saturated or may contain one or more units ofunsaturation, but which is not aromatic. Unless otherwise specified,aliphatic groups contain 1-12 carbon atoms. In some embodiments,aliphatic groups contain 1-6 carbon atoms. In some embodiments,aliphatic groups contain 1-4 carbon atoms, and in yet other embodimentsaliphatic groups contain 1-3 carbon atoms. Suitable aliphatic groupsinclude, but are not limited to, linear or branched, alkyl, alkenyl, andalkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl,(cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

Alkenyl—As used herein, the term “alkenyl” denotes an optionallysubstituted monovalent group derived from a straight- or branched-chainaliphatic moiety having at least one carbon-carbon double bond by theremoval of a single hydrogen atom. In particular embodiments, thealkenyl group employed in the invention contains 2-6 carbon atoms. Inparticular embodiments, the alkenyl group employed in the inventioncontains 2-5 carbon atoms. In some embodiments, the alkenyl groupemployed in the invention contains 2-4 carbon atoms. In anotherembodiment, the alkenyl group employed contains 2-3 carbon atoms.Alkenyl groups include, for example, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, and the like.

Alkyl—As used herein, the term “alkyl” refers to optionally substitutedsaturated, straight- or branched-chain hydrocarbon radicals derived froman aliphatic moiety containing between 1-6 carbon atoms by removal of asingle hydrogen atom. In some embodiments, the alkyl group employed inthe invention contains 1-5 carbon atoms. In another embodiment, thealkyl group employed contains 1-4 carbon atoms. In still otherembodiments, the alkyl group contains 1-3 carbon atoms. In yet anotherembodiment, the alkyl group contains 1-2 carbons. Examples of alkylradicals include, but are not limited to, methyl, ethyl, n-propyl,isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl,tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl,n-decyl, n-undecyl, dodecyl, and the like.

Alkynyl—As used herein, the term “alkynyl” refers to an optionallysubstituted monovalent group derived from a straight- or branched-chainaliphatic moiety having at least one carbon-carbon triple bond by theremoval of a single hydrogen atom. In particular embodiments, thealkynyl group employed in the invention contains 2-6 carbon atoms. Inparticular embodiments, the alkynyl group employed in the inventioncontains 2-5 carbon atoms. In some embodiments, the alkynyl groupemployed in the invention contains 2-4 carbon atoms. In anotherembodiment, the alkynyl group employed contains 2-3 carbon atoms.Representative alkynyl groups include, but are not limited to, ethynyl,2-propynyl (propargyl), 1-propynyl, and the like.

Aryl—As used herein, the term “aryl” used alone or as part of a largermoiety as in “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to anoptionally substituted monocyclic and bicyclic ring systems having atotal of five to 10 ring members, wherein at least one ring in thesystem is aromatic and wherein each ring in the system contains three toseven ring members. The term “aryl” may be used interchangeably with theterm “aryl ring”. In particular embodiments of the present invention,“aryl” refers to an aromatic ring system which includes, but not limitedto, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bearone or more substituents.

Arylalkyl—As used herein, the term “arylalkyl” refers to an alkyl groupsubstituted with an aryl group (e.g., an aromatic or heteroaromaticgroup).

Bidentate—a molecule formed from two or more molecules covalently boundtogether as a single unitmolecule.

Bivalent hydrocarbon chain—As used herein, the term “bivalenthydrocarbon chain” (also referred to as a “bivalent alkylene group”) isa polymethylene group, i.e., —(CH₂)_(z)—, wherein z is a positiveinteger from 1 to 30, from 1 to 20, from 1 to 12, from 1 to 8, from 1 to6, from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 30, from 2 to 20,from 2 to 10, from 2 to 8, from 2 to 6, from 2 to 4, or from 2 to 3. Asubstituted bivalent hydrocarbon chain is a polymethylene group in whichone or more methylene hydrogen atoms are replaced with a substituent.Suitable substituents include those described below for a substitutedaliphatic group.

Carbonyl—As used herein, the term “carbonyl” refers to a monovalent orbivalent moiety containing a carbon-oxygen double bond. Non-limitingexamples of carbonyl groups include aldehydes, ketones, carboxylicacids, ester, amide, enones, acyl halides, anhydrides, ureas,carbamates, carbonates, thioesters, lactones, lactams, hydroxamates,isocyanates, and chloroformates.

Cycloaliphatic—As used herein, the terms “cycloaliphatic”, “carbocycle”,or “carbocyclic”, used alone or as part of a larger moiety, refer to anoptionally substituted saturated or partially unsaturated cyclicaliphatic monocyclic or bicyclic ring systems, as described herein,having from 3 to 10 members. Cycloaliphatic groups include, withoutlimitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl,cyclooctenyl, and cyclooctadienyl. In some embodiments, the cycloalkylhas 3-6 carbons.

Fucose—refers to the D or L form of fucose and may refer to an oxygen orcarbon linked glycoside.

Halogen—As used herein, the terms “halo” and “halogen” refer to an atomselected from fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine(bromo, —Br), and iodine (iodo, —I).

Heteroaliphatic—As used herein, the terms “heteroaliphatic” or“heteroaliphatic group”, denote an optionally substituted hydrocarbonmoiety having, in addition to carbon atoms, from one to fiveheteroatoms, that may be straight-chain (i.e., unbranched), branched, orcyclic (“heterocyclic”) and may be completely saturated or may containone or more units of unsaturation, but which is not aromatic. Unlessotherwise specified, heteroaliphatic groups contain 1-6 carbon atomswherein 1-3 carbon atoms are optionally and independently replaced withheteroatoms selected from oxygen, nitrogen and sulfur. In someembodiments, heteroaliphatic groups contain 1-4 carbon atoms, wherein1-2 carbon atoms are optionally and independently replaced withheteroatoms selected from oxygen, nitrogen and sulfur. In yet otherembodiments, heteroaliphatic groups contain 1-3 carbon atoms, wherein 1carbon atom is optionally and independently replaced with a heteroatomselected from oxygen, nitrogen and sulfur. Suitable heteroaliphaticgroups include, but are not limited to, linear or branched, heteroalkyl,heteroalkenyl, and heteroalkynyl groups.

Heteroaralkyl—As used herein, the term “heteroaralkyl” refers to analkyl group substituted by a heteroaryl, wherein the alkyl andheteroaryl portions independently are optionally substituted.

Heteroaryl—As used herein, the term “heteroaryl” used alone or as partof a larger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refersto an optionally substituted group having 5 to 10 ring atoms, preferably5, 6, or 9 ring atoms; having 6, 10, or 14 π electrons shared in acyclic array; and having, in addition to carbon atoms, from one to fiveheteroatoms. Heteroaryl groups include, without limitation, thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and“heteroar-”, as used herein, also include groups in which aheteroaromatic ring is fused to one or more aryl, carbocyclic, orheterocyclic rings, where the radical or point of attachment is on theheteroaromatic ring. Non limiting examples include indolyl, isoindolyl,benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, andtetrahydroisoquinolinyl. A heteroaryl group may be mono- or bicyclic.The term “heteroaryl” may be used interchangeably with the terms“heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of whichterms include rings that are optionally substituted.

Heteroatom—As used herein, the term “heteroatom” refers to nitrogen,oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur,and any quaternized form of a basic nitrogen. The term “nitrogen” alsoincludes a substituted nitrogen.

Heterocyclic—As used herein, the terms “heterocycle”, “heterocyclyl”,“heterocyclic radical”, and “heterocyclic ring” are used interchangeablyand refer to a stable optionally substituted 5- to 7-membered monocyclicor 7 - to 10-membered bicyclic heterocyclic moiety that is eithersaturated or partially unsaturated, and having, in addition to carbonatoms, one or more heteroatoms, as defined above. A heterocyclic ringcan be attached to its pendant group at any heteroatom or carbon atomthat results in a stable structure and any of the ring atoms can beoptionally substituted. Examples of such saturated or partiallyunsaturated heterocyclic radicals include, without limitation,tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. Theterms “heterocycle”, “heterocyclyl”, “heterocyclyl ring”, “heterocyclicgroup”, “heterocyclic moiety”, and “heterocyclic radical”, are usedinterchangeably herein, and also include groups in which a heterocyclylring is fused to one or more aryl, heteroaryl, or carbocyclic rings,such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl, where the radical or point of attachment is on theheterocyclyl ring. A heterocyclyl group may be mono- or bicyclic. Theterm “heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

Unsaturated—As used herein, the term “unsaturated”, means that a moietyhas one or more double or triple bonds.

Partially unsaturated—As used herein, the term “partially unsaturated”refers to a ring moiety that includes at least one double or triplebond. The term “partially unsaturated” is intended to encompass ringshaving multiple sites of unsaturation, but is not intended to includearyl or heteroaryl moieties, as herein defined.

Optionally substituted —As described herein, compounds of the inventionmay contain “optionally substituted” moieties. In general, the term“substituted”, whether preceded by the term “optionally” or not, meansthat one or more hydrogens of the designated moiety are replaced with asuitable substituent. Unless otherwise indicated, an “optionallysubstituted” group may have a suitable substituent at each substitutableposition of the group, and when more than one position in any givenstructure may be substituted with more than one substituent selectedfrom a specified group, the substituent may be either the same ordifferent at every position. Combinations of substituents envisioned bythis invention are preferably those that result in the formation ofstable or chemically feasible compounds. The term “stable”, as usedherein, refers to compounds that are not substantially altered whensubjected to conditions to allow for their production, detection, and,in particular embodiments, their recovery, purification, and use for oneor more of the purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(∘); —(CH₂)₀₋₄OR^(∘); —O—(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄CH(OR^(∘))₂; —(CH₂)₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which may besubstituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(∘); —CH═CHPh, which may be substituted with R^(∘); —NO₂; —CN;—N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘); —N(R^(∘))C(S)R^(∘);—(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘) ₂;—(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘)) C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄OC(O)R^(∘);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(∘); —(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR^(∘)₂; —C(S)NR^(∘) ₂; —C(S)SR^(∘); —SC(S)SR^(∘), —(CH₂)₀₋₄OC(O)NR^(∘) ₂;—C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘); —C(O)CH₂C(O)R^(∘);—C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘); —(CH₂)₀₋₄S(O)₂R^(∘);—(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘); —S(O)₂NR^(∘) ₂;—(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂; —N(R^(∘))S(O)₂R^(∘);—N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘); —P(O)R^(∘) ₂; —OP(O)R^(∘)₂; —OP(O)(OR^(∘))₂; SiR^(∘) ₃; —(C₁₋₄ straight orbranched)alkylene)O—N(R^(∘))₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(R^(∘))₂, wherein each R^(∘)may be substituted asdefined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, or, not with standing the definition above, twoindependent occurrences of R^(∘), taken together with their interveningatom(s), form a 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, which may be substituted as definedbelow.

Suitable monovalent substituents on R^(∘)(or the ring formed by takingtwo independent occurrences of R^(∘)together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(haloR^(●)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●),—(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●),—(CH₂)₀₋₂NR^(●) ₂, —NO₂, —SiR^(●) ₃, —OSiR^(●) ₃, —C(O)SR^(●), —(C₁₋₄straight or branched alkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(∘)include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein each R^(●)isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or, not withstanding the definition above, two independent occurrences of R^(†),taken together with their intervening atom(s) form an unsubstituted3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN,—C(O)OH, —C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein eachR^(●) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable protecting group—As used herein, the term “suitable protectinggroup,” refers to amino protecting groups or hydroxyl protecting groupsdepending on its location within the compound and includes thosedescribed in detail in Protecting Groups in Organic Synthesis, T. W.Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999.

Suitable amino-protecting groups include methyl carbamate, ethylcarbamante, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′-and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative,N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonylderivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxycarbonylvinyl carbamate, o-N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate,formamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copperchelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.Suitable hydroxyl protecting groups include methyl, methoxylmethyl(MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide,1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP),1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts). For protecting 1,2- or 1,3-diols, the protecting groups includemethylene acetal, ethylidene acetal, 1-t-butylethylidene ketal,1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal,2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal,cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal,p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal,3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal,methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethyleneortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine orthoester, 1,2-dimethoxyethylidene ortho ester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivative,α-(N,N′-dimethylamino)benzylidene derivative, 2-oxacyclopentylideneortho ester, di-t-butylsilylene group (DTBS),1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cycliccarbonates, cyclic boronates, ethyl boronate, and phenyl boronate.

In any case where a chemical variable (e.g., an R group) is shownattached to a bond that crosses a bond of the ring, this means that oneor more such variables are optionally attached to the ring having thecrossed bond. Each R group on such a ring can be attached at anysuitable position on the ring, this is generally understood to mean thatthe group is attached in place of a hydrogen atom on the parent ring.This includes the possibility that two R groups can be attached to thesame ring atom. Furthermore, when more than one R group is present on aring, each may be the same or different than other R groups attachedthereto, and each group is defined independently of other groups thatmay be attached elsewhere on the same molecule, even though they may berepresented by the same identifier.

Biodegradable—As used herein, the term “biodegradable” refers tomolecules that degrade (i.e., lose at least some of their covalentstructure) under physiological or endosomal conditions. Biodegradablemolecules are not necessarily hydrolytically degradable and may requireenzymatic action to degrade.

Biomolecule—As used herein, the term “biomolecule” refers to molecules(e.g., polypeptides, amino acids, polynucleotides, nucleotides,polysaccharides, sugars, lipids, nucleoproteins, glycoproteins,lipoproteins, steroids, metabolites, etc.) whether naturally-occurringor artificially created (e.g., by synthetic or recombinant methods) thatare commonly found in cells and tissues. Specific classes ofbiomolecules include, but are not limited to, enzymes, receptors,neurotransmitters, hormones, cytokines, cell response modifiers such asgrowth factors and chemotactic factors, antibodies, vaccines, haptens,toxins, interferons, ribozymes, anti-sense agents, plasmids, DNA, andRNA.

Drug—As used herein, the term “drug” refers to small molecules orbiomolecules that alter, inhibit, activate, or otherwise affect abiological event. For example, drugs may include, but are not limitedto, anti-AIDS substances, anti-cancer substances, antibiotics,anti-diabetic substances, immunosuppressants, anti-viral substances,enzyme inhibitors, neurotoxins, opioids, hypnotics, anti-histamines,lubricants, tranquilizers, anti-convulsants, muscle relaxants andanti-Parkinson substances, anti-spasmodics and muscle contractantsincluding channel blockers, miotics and anti-cholinergics, anti-glaucomacompounds, anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, anti-secretory factors, anticoagulants and/or anti-thromboticagents, local anesthetics, ophthalmics, prostaglandins,anti-depressants, anti-psychotic substances, anti-emetics, and imagingagents. A more complete listing of exemplary drugs suitable for use inthe present invention may be found in “Pharmaceutical Substances:Syntheses, Patents, Applications” by Axel Kleemann and Jurgen Engel,Thieme Medical Publishing, 1999; the “Merck Index: An Encyclopedia ofChemicals, Drugs, and Biologicals”, edited by Susan Budavari et al., CRCPress, 1996, and the United States Pharmacopeia-25/NationalFormulary-20, published by the United States Pharmcopeial Convention,Inc., Rockville Md., 2001.

Exogenous—As used herein, an “exogenous” molecule is one which is notpresent at significant levels in a patient unless administered to thepatient. In particular embodiments the patient is a mammal, e.g., ahuman, a dog, a cat, a rat, a minipig, etc. As used herein, a moleculeis not present at significant levels in a patient if normal serum forthat type of patient includes less than 0.1 mM of the molecule. Inparticular embodiments, normal serum for the patient may include lessthan 0.08 mM, less than 0.06 mM, or less than 0.04 mM of the molecule.

Hyperbranched—As used herein, a “hyperbranched” structure is a covalentstructure that includes at least one branched branch (e.g., adendrimeric structure). A hyperbranched structure may include polymericand/or non-polymeric substructures. Normal serum—As used herein, “normalserum” is serum obtained by pooling approximately equal amounts of theliquid portion of coagulated whole blood from five or more non-diabeticpatients. A non-diabetic human patient is a randomly selected 18-30 yearold who presents with no diabetic symptoms at the time blood is drawn.

Polymer—As used herein, a “polymer” or “polymeric structure” is astructure that includes a string of covalently bound monomers. A polymercan be made from one type of monomer or more than one type of monomer.The term “polymer” therefore encompasses copolymers, includingblock-copolymers in which different types of monomer are groupedseparately within the overall polymer. A polymer can be linear orbranched.

Polynucleotide—As used herein, a “polynucleotide” is a polymer ofnucleotides. The terms “polynucleotide”, “nucleic acid”, and“oligonucleotide” may be used interchangeably. The polymer may includenatural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine,uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, anddeoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,5-methylcytidine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine,dihydrouridine, methylpseudouridine, 1-methyl adenosine, 1-methylguanosine, N6-methyl adenosine, and 2-thiocytidine), chemically modifiedbases, biologically modified bases (e.g., methylated bases),intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose,2′-deoxyribose, 2′-O-methylcytidine, arabinose, and hexose), or modifiedphosphate groups (e.g., phosphorothioates and 5′-N-phosphoramiditelinkages).

Polypeptide—As used herein, a “polypeptide” is a polymer of amino acids.The terms “polypeptide”, “protein”, “oligopeptide”, and “peptide” may beused interchangeably. Polypeptides may contain natural amino acids,non-natural amino acids (i.e., compounds that do not occur in nature butthat can be incorporated into a polypeptide chain) and/or amino acidanalogs as are known in the art. Also, one or more of the amino acidresidues in a polypeptide may be modified, for example, by the additionof a chemical entity such as a carbohydrate group, a phosphate group, afarnesyl group, an isofarnesyl group, a fatty acid group, a linker forconjugation, functionalization, or other modification, etc. Thesemodifications may include cyclization of the peptide, the incorporationof D-amino acids, etc.

Polysaccharide—As used herein, a “polysaccharide” is a polymer ofsaccharides. The terms “polysaccharide”, “carbohydrate”, and“oligosaccharide”, may be used interchangeably. The polymer may includenatural saccharides (e.g., arabinose, lyxose, ribose, xylose, ribulose,xylulose, allose, altrose, galactose, glucose, gulose, idose, mannose,talose, fructose, psicose, sorbose, tagatose, mannoheptulose,sedoheptulose, octolose, and sialose) and/or modified saccharides (e.g.,2′-fluororibose, 2′-deoxyribose, and hexose). Exemplary disaccharidesinclude sucrose, lactose, maltose, trehalose, gentiobiose, isomaltose,kojibiose, laminaribiose, mannobiose, melibiose, nigerose, rutinose, andxylobiose.

Small molecule—As used herein, the term “small molecule” refers tomolecules, whether naturally-occurring or artificially created (e.g.,via chemical synthesis), that have a relatively low molecular weight.Typically, small molecules are monomeric and have a molecular weight ofless than about 1500 Da. Preferred small molecules are biologicallyactive in that they produce a local or systemic effect in animals,preferably mammals, more preferably humans. In particular preferredembodiments, the small molecule is a drug. Preferably, though notnecessarily, the drug is one that has already been deemed safe andeffective for use by the appropriate governmental agency or body. Forexample, drugs for human use listed by the FDA under 21 C.F.R. §§330.5,331 through 361, and 440 through 460; drugs for veterinary use listed bythe FDA under 21 C.F.R. §§500 through 589, are all considered acceptablefor use in accordance with the present invention.

Treat—As used herein, the term “treat” (or “treating”, “treated”,“treatment”, etc.) refers to the administration of a conjugate of thepresent disclosure to a subject in need thereof with the purpose toalleviate, relieve, alter, ameliorate, improve or affect a condition(e.g., diabetes), a symptom or symptoms of a condition (e.g.,hyperglycemia), or the predisposition toward a condition. For example,as used herein the term “treating diabetes” will refer in general tomaintaining glucose blood levels near normal levels and may includeincreasing or decreasing blood glucose levels depending on a givensituation.

Pharmaceutically acceptable carrier—as used herein, the term includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

Pharmaceutically acceptable salt—as used herein, the term refers tosalts of compounds that retain the biological activity of the parentcompound, and which are not biologically or otherwise undesirable. Manyof the compounds disclosed herein are capable of forming acid and/orbase salts by virtue of the presence of amino and/or carboxyl groups orgroups similar thereto.

Pharmaceutically acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

Effective or therapeutically effective amount—as used herein refers to anontoxic but sufficient amount of an insulin analog to provide thedesired effect. For example one desired effect would be the preventionor treatment of hyperglycemia. The amount that is “effective” will varyfrom subject to subject, depending on the age and general condition ofthe individual, mode of administration, and the like. Thus, it is notalways possible to specify an exact “effective amount.” However, anappropriate “effective” amount in any individual case may be determinedby one of ordinary skill in the art using routine experimentation.

Parenteral—as used herein, the term means not through the alimentarycanal but by some other route such as intranasal, inhalation,subcutaneous, intramuscular, intraspinal, or intravenous.

Insulin—as used herein, the term means the active principle of thepancreas that affects the metabolism of carbohydrates in the animal bodyand which is of value in the treatment of diabetes mellitus. The termincludes synthetic and biotechnologically derived products that are thesame as, or similar to, naturally occurring insulins in structure, use,and intended effect and are of value in the treatment of diabetesmellitus.

Insulin or insulin molecule—the term is a generic term that designatesthe 51 amino acid heterodimer comprising the A-chain peptide having theamino acid sequence shown in SEQ ID NO: 1 and the B-chain peptide havingthe amino acid sequence shown in SEQ ID NO: 2, wherein the cysteineresidues a positions 6 and 11 of the A chain are linked in a disulfidebond, the cysteine residues at position 7 of the A chain and position 7of the B chain are linked in a disulfide bond, and the cysteine residuesat position 20 of the A chain and 19 of the B chain are linked in adisulfide bond.

Insulin analog or analogue—the term as used herein includes anyheterodimer analogue or single-chain analogue that comprises one or moremodification(s) of the native A-chain peptide and/or B-chain peptide.Modifications include but are not limited to substituting an amino acidfor the native amino acid at a position selected from A4, A5, A8, A9,A10, A12, A13, A14, A15, A16, A17, A18, A19, A21, B1, B2, B3, B4, B5,B9, B10, B13, B14, B15, B16, B17, B18, B20, B21, B22, B23, B26, B27,B28, B29, and B30; deleting any or all of positions B1-4 and B26-30; orconjugating directly or by a polymeric or non-polymeric linker one ormore acyl, polyethylglycine (PEG), or saccharide moiety (moieties); orany combination thereof. As exemplified by the N-linked glycosylatedinsulin analogues disclosed herein, the term further includes anyinsulin heterodimer and single-chain analogue that has been modified tohave at least one N-linked glycosylation site and in particular,embodiments in which the N-linked glycosylation site is linked to oroccupied by an N-glycan. Examples of insulin analogues include but arenot limited to the heterodimer and single-chain analogues disclosed inpublished international application WO20100080606, WO2009/099763, andWO2010080609, the disclosures of which are incorporated herein byreference. Examples of single-chain insulin analogues also include butare not limited to those disclosed in published InternationalApplications WO9634882, WO95516708, WO2005054291, WO2006097521,WO2007104734, WO2007104736, WO2007104737, WO2007104738, WO2007096332,WO2009132129; U.S. Pat. Nos. 5,304,473 and 6,630,348; and Kristensen etal., Biochem. J. 305: 981-986 (1995), the disclosures of which are eachincorporated herein by reference.

The term further includes single-chain and heterodimer polypeptidemolecules that have little or no detectable activity at the insulinreceptor but which have been modified to include one or more amino acidmodifications or substitutions to have an activity at the insulinreceptor that has at least 1%, 10%, 50%, 75%, or 90% of the activity atthe insulin receptor as compared to native insulin and which furtherincludes at least one N-linked glycosylation site. In particularaspects, the insulin analogue is a partial agonist that has from 2× to100× less activity at the insulin receptor as does native insulin. Inother aspects, the insulin analogue has enhanced activity at the insulinreceptor, for example, the IGF^(B16B17) derivative peptides disclosed inpublished international application WO2010080607 (which is incorporatedherein by reference). These insulin analogues, which have reducedactivity at the insulin growth hormone receptor and enhanced activity atthe insulin receptor, include both heterodimers and single-chainanalogues.

Single-chain insulin or single-chain insulin analog—as used herein, theterm encompasses a group of structurally-related proteins wherein theA-chain peptide or functional analogue and the B-chain peptide orfunctional analogue are covalently linked by a peptide or polypeptide of2 to 35 amino acids or non-peptide polymeric or non-polymeric linker andwhich has at least 1%, 10%, 50%, 75%, or 90% of the activity of insulinat the insulin receptor as compared to native insulin. The single-chaininsulin or insulin analogue further includes three disulfide bonds: thefirst disulfide bond is between the cysteine residues at positions 6 and11 of the A-chain or functional analogue thereof, the second disulfidebond is between the cysteine residues at position 7 of the A-chain orfunctional analogue thereof and position 7 of the B-chain or functionalanalogue thereof, and the third disulfide bond is between the cysteineresidues at position 20 of the A-chain or functional analogue thereofand position 19 of the B-chain or functional analogue thereof.

Connecting peptide or C-peptide—as used herein, the term refers to theconnection moiety “C” of the B-C-A polypeptide sequence of a singlechain preproinsulin-like molecule. Specifically, in the natural insulinchain, the C-peptide connects the amino acid at position 30 of theB-chain and the amino acid at position 1 of the A-chain. The term canrefer to both the native insulin C-peptide (SEQ ID NO:30), the monkeyC-peptide, and any other peptide from 3 to 35 amino acids that connectsthe B-chain to the A-chain thus is meant to encompass any peptidelinking the B-chain peptide to the A-chain peptide in a single-chaininsulin analogue (See for example, U.S. Published application Nos.20090170750 and 20080057004 and WO9634882) and in insulin precursormolecules such as disclosed in WO9516708 and U.S. Pat. No. 7,105,314.

Amino acid modification—as used herein, the term refers to asubstitution of an amino acid, or the derivation of an amino acid by theaddition and/or removal of chemical groups to/from the amino acid, andincludes substitution with any of the 20 amino acids commonly found inhuman proteins, as well as a typical or non-naturally occurring aminoacids. Commercial sources of a typical amino acids include Sigma-Aldrich(Milwaukee, Wis.), ChemPep Inc. (Miami, Fla.), and GenzymePharmaceuticals (Cambridge, Mass.). Atypical amino acids may bepurchased from commercial suppliers, synthesized de novo, or chemicallymodified or derivatized from naturally occurring amino acids.

-   -   Amino acid substitution—as used herein refers to the replacement        of one amino acid residue by a different amino acid residue.    -   Conservative amino acid substitution—as used herein, the term is        defined herein as exchanges within one of the following five        groups:    -   I. Small aliphatic, nonpolar or slightly polar residues:        -   Ala, Ser, Thr, Pro, Gly;    -   II. Polar, negatively charged residues and their amides:        -   Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid;    -   III. Polar, positively charged residues:        -   His, Arg, Lys; Ornithine (Orn)    -   IV. Large, aliphatic, nonpolar residues:        -   Met, Leu, Ile, Val, Cys, Norleucine (Nle), homocysteine    -   V. Large, aromatic residues:        -   Phe, Tyr, Trp, acetyl phenylalanine

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: shows blood glucose depression curves in non-diabetic maleYucatan minipigs equipped with dual vascular access ports (n=3 perstudy) following i.v. injection of conjugate IOC-2 at 0.69 nmol/kg underconditions of PBS infusion or i.v. alpha methyl mannose (aMM) infusion.

FIG. 2: shows blood glucose depression curves in non-diabetic maleYucatan minipigs equipped with dual vascular access ports (n=3 perstudy) following i.v. injection of conjugate IOC-3 at 0.17 nmol/kg underconditions of PBS infusion or i.v. alpha methyl mannose (aMM) infusion.

FIG. 3: shows blood glucose depression curves in non-diabetic maleYucatan minipigs equipped with dual vascular access ports (n=3 perstudy) following i.v. injection of conjugate IOC-8 at 0.17 nmol/kg underconditions of PBS infusion or i.v. alpha methyl mannose (aMM) infusion.

FIG. 4: shows blood glucose depression curves in non-diabetic maleYucatan minipigs equipped with dual vascular access ports (n=3 perstudy) following i.v. injection of conjugate IOC-9 at 0.17 nmol/kg underconditions of PBS infusion or i.v. alpha methyl mannose (aMM) infusion.

FIG. 5: shows blood glucose depression curves in non-diabetic maleYucatan minipigs equipped with dual vascular access ports (n=3 perstudy) following i.v. injection of conjugate IOC-16 at 0.35 nmol/kgunder conditions of PBS infusion or i.v. alpha methyl mannose (aMM)infusion.

FIG. 6: shows blood glucose depression curves in non-diabetic maleYucatan minipigs equipped with dual vascular access ports (n=3 perstudy) following i.v. injection of conjugate IOC-22 at 0.35 nmol/kgunder conditions of PBS infusion or i.v. alpha methyl mannose (aMM)infusion.

FIG. 7: shows blood glucose depression curves in non-diabetic maleYucatan minipigs equipped with dual vascular access ports (n=3 perstudy) following i.v. injection of conjugate IOC-23 at 0.35 nmol/kgunder conditions of PBS infusion or i.v. alpha methyl mannose (aMM)infusion.

FIG. 8: shows blood glucose depression curves in non-diabetic maleYucatan minipigs equipped with dual vascular access ports (n=3 perstudy) following i.v. injection of conjugate IOC-46 at 0.35 nmol/kgunder conditions of PBS infusion or i.v. alpha methyl mannose (aMM)infusion.

FIG. 9: shows blood glucose depression curves in non-diabetic maleYucatan minipigs equipped with dual vascular access ports (n=3 perstudy) following i.v. injection of conjugate IOC-48 under conditions ofPBS infusion or i.v. alpha methyl mannose (aMM) infusion.

FIG. 10: shows blood glucose depression curves in non-diabetic maleYucatan minipigs equipped with dual vascular access ports (n=3 perstudy) following i.v. injection of conjugate IOC-52 at 0.35 nmol/kgunder conditions of PBS infusion or i.v. alpha methyl mannose (aMM)infusion.

FIG. 11: shows blood glucose depression curves in non-diabetic maleYucatan minipigs equipped with dual vascular access ports (n=3 perstudy) following i.v. injection of conjugate IOC-56 at 0.69 nmol/kgunder conditions of PBS infusion or i.v. alpha methyl mannose (aMM)infusion.

FIG. 12: shows blood glucose depression curves in non-diabetic maleYucatan minipigs equipped with dual vascular access ports (n=3 perstudy) following i.v. injection of conjugate IOC-60 at 0.35 nmol/kgunder conditions of PBS infusion or i.v. alpha methyl mannose (aMM)infusion.

FIG. 13: shows blood glucose depression curves in non-diabetic maleYucatan minipigs equipped with dual vascular access ports (n=3 perstudy) following i.v. injection of conjugate IOC-75 at 0.69 nmol/kgunder conditions of PBS infusion or i.v. alpha methyl mannose (aMM)infusion.

FIG. 14: shows blood glucose depression curves in non-diabetic maleYucatan minipigs equipped with dual vascular access ports (n=3 perstudy) following i.v. injection of conjugate IOC-76 at 0.17 nmol/kgunder conditions of PBS infusion or i.v. alpha methyl mannose (aMM)infusion.

FIG. 15: shows plots of serum concentrations of IOC-2 following a 0.69nmol/kg intravenous (i.v.) injection into non-diabetic male Yucatanminipigs equipped with dual vascular access ports (n=3 per study)infused with i.v. alpha methyl mannose (aMM) solution (21.2% w/v infusedat constant rate of 2.67 mL/kg/hr) or PBS.

FIG. 16: shows plots of serum concentrations of IOC-3 following a 0.17nmol/kg intravenous (i.v.) injection into non-diabetic male Yucatanminipigs equipped with dual vascular access ports (n=3 per study)infused with i.v. alpha methyl mannose (aMM) solution (21.2% w/v infusedat constant rate of 2.67 mL/kg/hr) or PBS.

FIG. 17: shows plots of serum concentrations of IOC-16 following a 0.35nmol/kg intravenous (i.v.) injection into non-diabetic male Yucatanminipigs equipped with dual vascular access ports (n=3 per study)infused with i.v. alpha methyl mannose (aMM) solution (21.2% w/v infusedat constant rate of 2.67 mL/kg/hr) or PBS.

FIG. 18: shows plots of serum concentrations of IOC-22 following a 0.35nmol/kg intravenous (i.v.) injection into non-diabetic male Yucatanminipigs equipped with dual vascular access ports (n=3 per study)infused with i.v. alpha methyl mannose (aMM) solution (21.2% w/v infusedat constant rate of 2.67 mL/kg/hr) or PBS.

FIG. 19: shows plots of serum concentrations of IOC-23 following a 0.35nmol/kg intravenous (i.v.) injection into non-diabetic male Yucatanminipigs equipped with dual vascular access ports (n=3 per study)infused with i.v. alpha methyl mannose (aMM) solution (21.2% w/v infusedat constant rate of 2.67 mL/kg/hr) or PBS.

FIG. 20: shows plots of serum concentrations of IOC-52 following a 0.35nmol/kg intravenous (i.v.) injection into non-diabetic male Yucatanminipigs equipped with dual vascular access ports (n=3 per study)infused with i.v. alpha methyl mannose (aMM) solution (21.2% w/v infusedat constant rate of 2.67 mL/kg/hr) or PBS.

FIG. 21: shows plots of serum concentrations of IOC-56 following a 0.69nmol/kg intravenous (i.v.) injection into non-diabetic male Yucatanminipigs equipped with dual vascular access ports (n=3 per study)infused with i.v. alpha methyl mannose (aMM) solution (21.2% w/v infusedat constant rate of 2.67 mL/kg/hr) or PBS.

FIG. 22: shows plots of serum concentrations of IOC-60 following a 0.35nmol/kg intravenous (i.v.) injection into non-diabetic male Yucatanminipigs equipped with dual vascular access ports (n=3 per study)infused with i.v. alpha methyl mannose (aMM) solution (21.2% w/v infusedat constant rate of 2.67 mL/kg/hr) or PBS.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for controlling thepharmacokinetic (PK) and/or pharmacodynamic (PD) profiles of insulin ina manner that is responsive to the systemic concentrations of asaccharide such as glucose. The methods are based in part on thediscovery disclosed in U.S. Published Application No. 2011/0301083 thatwhen particular insulin conjugates are modified to include high affinitysaccharide ligands such as branched trimannose, they could be made toexhibit PK/PD profiles that responded to saccharide concentrationchanges even in the absence of an exogenous multivalentsaccharide-binding molecule such as the lectin Concanavalin A (Con A).

In general, the insulin conjugates of the present invention comprise aninsulin or insulin analog molecule covalently attached to at least onebranched linker having or consisting of two arms, each arm independentlycovalently attached to a ligand comprising or consisting of a saccharidewherein at least one ligand of the linker includes the saccharidefucose. In particular embodiments, the ligands are capable of competingwith a saccharide (e.g., glucose or alpha-methylmannose) for binding toan endogenous saccharide-binding molecule. In particular embodiments,the ligands are capable of competing with glucose or alpha-methylmannosefor binding to Con A. In particular embodiments, the linker isnon-polymeric. In particular embodiments, the conjugate may have apolydispersity index of one and a MW of less than about 20,000 Da. Inparticular embodiments, the conjugate is of formula (I) or (II) asdefined and described herein. In particular embodiments, the conjugateis long acting (i.e., exhibits a PK profile that is more sustained thansoluble recombinant human insulin (RHI)).

As used herein, the term “insulin conjugate” includes (i) insulinconjugates comprising an insulin molecule have the native or wild-typeamino acid sequence of insulin and (ii) insulin conjugates comprising aninsulin analog molecule wherein the insulin analog comprises an aminoacid sequence that differs from the native or wild-type insulin aminoacid sequence by at least one amino acid substitution, deletion,rearrangement, or addition. The term further includes insulin or insulinanalog molecules that are conjugated to a polyethylene glycol or fattyacid molecule. The insulin molecule may be human insulin, porcineinsulin, bovine insulin, rabbit insulin, sheep insulin, etc. or analogthereof. A number of these insulin molecules are available commercially,e.g., from Sigma-Aldrich (St. Louis, Mo.). A variety of modified formsof insulin are known in the art (e.g., see Crotty and Reynolds, Pediatr.Emerg. Care. 23:903-905, 2007 and Gerich, Am. J. Med. 113:308-16, 2002and references cited therein). Modified forms of insulin may bechemically modified (e.g., by addition of a chemical moiety such as aPEG group or a fatty acyl chain as described below) and/or mutated(i.e., by addition, deletion or substitution of one or more aminoacids).

Insulin Conjugates

In one aspect, the present invention provides insulin conjugates thatcomprise an insulin or insulin analog molecule covalently attached to atleast one branched linker having two arms (bi-dentate linker) whereineach arm of the bi-dentate linker is independently covalently linked toa ligand comprising or consisting of a saccharide and wherein the firstligand of the bi-dentate linker comprises or consists of a firstsaccharide, which is fucose. The second ligand of the bi-dentate linkercomprises or consists of a second saccharide, which may be fucose,mannose, glucosamine, or glucose. In particular aspects, the secondligand comprises or consists of a bisaccharide, trisaccharide,tetrasaccharide, or branched trisaccharide. In particular aspects, thesecond ligand comprises a bimannose, trimannose, tetramannose, orbranched trimannose.

In particular aspects, the insulin or insulin analog molecule isconjugated to one, two, three, or four bi-dentate linkers wherein eacharm of each bi-dentate linker is independently covalently linked to aligand comprising or consisting of a saccharide and wherein the firstligand of the bi-dentate linker comprises or consists of a firstsaccharide, which is fucose, and the second ligand of the bi-dentatelinker comprises or consists of a second saccharide, which may befucose, mannose, or glucose. In particular aspects, the second ligandcomprises or consists of a bisaccharide, trisaccharide, tetrasaccharide,or branched trisaccharide. In particular aspects, the second ligandcomprises or consists of a bimannose, trimannose, tetramannose, orbranched trimannose.

In particular aspects, the insulin or insulin analog molecule isconjugated to one, two, three, or four bi-dentate linkers wherein eacharm of each bi-dentate linker is independently covalently linked to aligand comprising or consisting of a saccharide and wherein for at leastone of the bi-dentate linkers the first ligand of the bi-dentate linkercomprises or consists of a first saccharide, which is fucose, and thesecond ligand of the bi-dentate linker comprises or consists of a secondsaccharide, which may be fucose, mannose, or glucose. In particularaspects, the second ligand comprises or consists of a bisaccharide,trisaccharide, tetrasaccharide, or branched trisaccharide. In particularaspects, the second ligand comprises or consists of a bimannose,trimannose, tetramannose, or branched trimannose. For the second, third,and fourth bi-dentate linkers, the first and second saccharides mayindependently be fucose, mannose, glucose, bisaccharide, trisaccharide,tetrasaccharide, branched trisaccharide, bimannose, trimannose,tetramannose, or branched trimannose.

In particular aspects, the insulin or insulin analog molecule isconjugated to (i) one bi-dentate linker wherein each arm of eachbi-dentate linker is independently covalently linked to a ligandcomprising or consisting of a saccharide wherein the first ligand of thebi-dentate linker comprises or consists of a first saccharide, which isfucose, and the second ligand of the bi-dentate linker comprises orconsists of a second saccharide, which may be fucose, mannose, glucose,bisaccharide, trisaccharide, tetrasaccharide, branched trisaccharide,bimannose, trimannose, tetramannose, or branched trimannose.

In particular aspects, the insulin or insulin analog molecule of theinsulin conjugate disclosed herein is further covalently attached to atleast one linear linker having one ligand comprising or consisting of asaccharide, which may be fucose, mannose, glucosamine, or glucose. Inparticular aspects, the ligand comprises or consisting of abisaccharide, trisaccharide, tetrasaccharide, or branched trisaccharide.In particular aspects, the ligand comprises or consisting of abimannose, trimannose, tetramannose, or branched trimannose.

In particular aspects, the insulin or insulin analog molecule conjugatedisclosed herein is further covalently attached to at least onetri-dentate linker wherein each arm of the tri-dentate linker isindependently covalently linked to a ligand comprising or consisting ofa saccharide, which may be fucose, mannose, glucosamine, or glucose. Inparticular aspects, the ligand comprises or consisting of abisaccharide, trisaccharide, tetrasaccharide, or branched trisaccharide.In particular aspects, the ligand comprises or consisting of abimannose, trimannose, tetramannose, or branched trimannose.

When the insulin conjugate is administered to a mammal at least onepharmacokinetic or pharmacodynamic property of the conjugate issensitive to the serum concentration of a saccharide. In particularembodiments, the PK and/or PD properties of the conjugate are sensitiveto the serum concentration of an endogenous saccharide such as glucose.In particular embodiments, the PK and/or PD properties of the conjugateare sensitive to the serum concentration of an exogenous saccharide,e.g., without limitation, mannose, L-fucose, N-acetyl glucosamine and/oralpha-methyl mannose.

PK and PD Properties

In various embodiments, the pharmacokinetic and/or pharmacodynamicbehavior of the insulin conjugate may be modified by variations in theserum concentration of a saccharide. For example, from a pharmacokinetic(PK) perspective, the serum concentration curve may shift upward whenthe serum concentration of the saccharide (e.g., glucose) increases orwhen the serum concentration of the saccharide crosses a threshold(e.g., is higher than normal glucose levels).

In particular embodiments, the serum concentration curve of a conjugateis substantially different when administered to the mammal under fastedand hyperglycemic conditions. As used herein, the term “substantiallydifferent” means that the two curves are statistically different asdetermined by a student t-test (p<0.05). As used herein, the term“fasted conditions” means that the serum concentration curve wasobtained by combining data from five or more fasted non-diabeticindividuals. In particular embodiments, a fasted non-diabetic individualis a randomly selected 18-30 year old human who presents with nodiabetic symptoms at the time blood is drawn and who has not eatenwithin 12 hours of the time blood is drawn. As used herein, the term“hyperglycemic conditions” means that the serum concentration curve wasobtained by combining data from five or more fasted non-diabeticindividuals in which hyperglycemic conditions (glucose C_(max) at least100 mg/dL above the mean glucose concentration observed under fastedconditions) were induced by concurrent administration of conjugate andglucose. Concurrent administration of conjugate and glucose simplyrequires that the glucose C_(max) occur during the period when theconjugate is present at a detectable level in the serum. For example, aglucose injection (or ingestion) could be timed to occur shortly before,at the same time or shortly after the conjugate is administered. Inparticular embodiments, the conjugate and glucose are administered bydifferent routes or at different locations. For example, in particularembodiments, the conjugate is administered subcutaneously while glucoseis administered orally or intravenously.

In particular embodiments, the serum C_(max) of the conjugate is higherunder hyperglycemic conditions as compared to fasted conditions.Additionally or alternatively, in particular embodiments, the serum areaunder the curve (AUC) of the conjugate is higher under hyperglycemicconditions as compared to fasted conditions. In various embodiments, theserum elimination rate of the conjugate is slower under hyperglycemicconditions as compared to fasted conditions. In particular embodiments,the serum concentration curve of the conjugates can be fit using atwo-compartment bi-exponential model with one short and one longhalf-life. The long half-life appears to be particularly sensitive toglucose concentration. Thus, in particular embodiments, the longhalf-life is longer under hyperglycemic conditions as compared to fastedconditions. In particular embodiments, the fasted conditions involve aglucose C_(max) of less than 100 mg/dL (e.g., 80 mg/dL, 70 mg/dL, 60mg/dL, 50 mg/dL, etc.). In particular embodiments, the hyperglycemicconditions involve a glucose C_(max) in excess of 200 mg/dL (e.g., 300mg/dL, 400 mg/dL, 500 mg/dL, 600 mg/dL, etc.). It will be appreciatedthat other PK parameters such as mean serum residence time (MRT), meanserum absorption time (MAT), etc. could be used instead of or inconjunction with any of the aforementioned parameters.

The normal range of glucose concentrations in humans, dogs, cats, andrats is 60 to 200 mg/dL. One skilled in the art will be able toextrapolate the following values for species with different normalranges (e.g., the normal range of glucose concentrations in miniaturepigs is 40 to 150 mg/dl). Glucose concentrations below 60 mg/dL areconsidered hypoglycemic. Glucose concentrations above 200 mg/dL areconsidered hyperglycemic. In particular embodiments, the PK propertiesof the conjugate may be tested using a glucose clamp method (seeExamples) and the serum concentration curve of the conjugate may besubstantially different when administered at glucose concentrations of50 and 200 mg/dL, 50 and 300 mg/dL, 50 and 400 mg/dL, 50 and 500 mg/dL,50 and 600 mg/dL, 100 and 200 mg/dL, 100 and 300 mg/dL, 100 and 400mg/dL, 100 and 500 mg/dL, 100 and 600 mg/dL, 200 and 300 mg/dL, 200 and400 mg/dL, 200 and 500 mg/dL, 200 and 600 mg/dL, etc. Additionally oralternatively, the serum T_(max), serum C_(max), mean serum residencetime (MRT), mean serum absorption time (MAT) and/or serum half-life maybe substantially different at the two glucose concentrations. Asdiscussed below, in particular embodiments, 100 mg/dL and 300 mg/dL maybe used as comparative glucose concentrations. It is to be understoodhowever that the present disclosure encompasses each of theseembodiments with an alternative pair of comparative glucoseconcentrations including, without limitation, any one of the followingpairs: 50 and 200 mg/dL, 50 and 300 mg/dL, 50 and 400 mg/dL, 50 and 500mg/dL, 50 and 600 mg/dL, 100 and 200 mg/dL, 100 and 400 mg/dL, 100 and500 mg/dL, 100 and 600 mg/dL, 200 and 300 mg/dL, 200 and 400 mg/dL, 200and 500 mg/dL, 200 and 600 mg/dL, etc.

Thus, in particular embodiments, the C_(max) of the conjugate is higherwhen administered to the mammal at the higher of the two glucoseconcentrations (e.g., 300 vs. 100 mg/dL glucose). In particularembodiments, the C_(max) of the conjugate is at least 50% (e.g., atleast 100%, at least 200% or at least 400%) higher when administered tothe mammal at the higher of the two glucose concentrations (e.g., 300vs. 100 mg/dL glucose).

In particular embodiments, the AUC of the conjugate is higher whenadministered to the mammal at the higher of the two glucoseconcentrations (e.g., 300 vs. 100 mg/dL glucose). In particularembodiments, the AUC of the conjugate is at least 50% (e.g., at leaste.g., at least 100%, at least 200% or at least 400%) higher whenadministered to the mammal at the higher of the two glucoseconcentrations (e.g., 300 vs. 100 mg/dL glucose).

In particular embodiments, the serum elimination rate of the conjugateis slower when administered to the mammal at the higher of the twoglucose concentrations (e.g., 300 vs. 100 mg/dL glucose). In particularembodiments, the serum elimination rate of the conjugate is at least 25%(e.g., at least 50%, at least 100%, at least 200%, or at least 400%)faster when administered to the mammal at the lower of the two glucoseconcentrations (e.g., 100 vs. 300 mg/dL glucose).

In particular embodiments the serum concentration curve of conjugatesmay be fit using a two-compartment bi-exponential model with one shortand one long half-life. The long half-life appears to be particularlysensitive to glucose concentration. Thus, in particular embodiments, thelong half-life is longer when administered to the mammal at the higherof the two glucose concentrations (e.g., 300 vs. 100 mg/dL glucose). Inparticular embodiments, the long half-life is at least 50% (e.g., atleast 100%, at least 200% or at least 400%) longer when administered tothe mammal at the higher of the two glucose concentrations (e.g., 300vs. 100 mg/dL glucose).

In particular embodiments, the present disclosure provides a method inwhich the serum concentration curve of a conjugate is obtained at twodifferent glucose concentrations (e.g., 300 vs. 100 mg/dL glucose); thetwo curves are fit using a two-compartment bi-exponential model with oneshort and one long half-life; and the long half-lives obtained under thetwo glucose concentrations are compared. In particular embodiments, thismethod may be used as an assay for testing or comparing the glucosesensitivity of one or more conjugates.

In particular embodiments, the present disclosure provides a method inwhich the serum concentration curves of a conjugated drug (e.g., aninsulin conjugate of the present disclosure) and an unconjugated versionof the drug (e.g., RHI) are obtained under the same conditions (e.g.,fasted conditions); the two curves are fit using a two-compartmentbi-exponential model with one short and one long half-life; and the longhalf-lives obtained for the conjugated and unconjugated drug arecompared. In particular embodiments, this method may be used as an assayfor identifying conjugates that are cleared more rapidly than theunconjugated drug.

In particular embodiments, the serum concentration curve of a conjugateis substantially the same as the serum concentration curve of anunconjugated version of the drug when administered to the mammal underhyperglycemic conditions. As used herein, the term “substantially thesame” means that there is no statistical difference between the twocurves as determined by a student t-test (p>0.05). In particularembodiments, the serum concentration curve of the conjugate issubstantially different from the serum concentration curve of anunconjugated version of the drug when administered under fastedconditions. In particular embodiments, the serum concentration curve ofthe conjugate is substantially the same as the serum concentration curveof an unconjugated version of the drug when administered underhyperglycemic conditions and substantially different when administeredunder fasted conditions.

In particular embodiments, the hyperglycemic conditions involve aglucose C_(max) in excess of 200 mg/dL (e.g., 300 mg/dL, 400 mg/dL, 500mg/dL, 600 mg/dL, etc.). In particular embodiments, the fastedconditions involve a glucose C_(max) of less than 100 mg/dL (e.g., 80mg/dL, 70 mg/dL, 60 mg/dL, 50 mg/dL, etc.). It will be appreciated thatany of the aforementioned PK parameters such as serum T_(max), serumC_(max), AUC, mean serum residence time (MRT), mean serum absorptiontime (MAT) and/or serum half-life could be compared. From apharmacodynamic (PD) perspective, the bioactivity of the conjugate mayincrease when the glucose concentration increases or when the glucoseconcentration crosses a threshold, e.g., is higher than normal glucoselevels. In particular embodiments, the bioactivity of a conjugate islower when administered under fasted conditions as compared tohyperglycemic conditions. In particular embodiments, the fastedconditions involve a glucose C_(max) of less than 100 mg/dL (e.g., 80mg/dL, 70 mg/dL, 60 mg/dL, 50 mg/dL, etc.). In particular embodiments,the hyperglycemic conditions involve a glucose C_(max) in excess of 200mg/dL (e.g., 300 mg/dL, 400 mg/dL, 500 mg/dL, 600 mg/dL, etc.).

In particular embodiments, the PD properties of the conjugate may betested by measuring the glucose infusion rate (GIR) required to maintaina steady glucose concentration. According to such embodiments, thebioactivity of the conjugate may be substantially different whenadministered at glucose concentrations of 50 and 200 mg/dL, 50 and 300mg/dL, 50 and 400 mg/dL, 50 and 500 mg/dL, 50 and 600 mg/dL, 100 and 200mg/dL, 100 and 300 mg/dL, 100 and 400 mg/dL, 100 and 500 mg/dL, 100 and600 mg/dL, 200 and 300 mg/dL, 200 and 400 mg/dL, 200 and 500 mg/dL, 200and 600 mg/dL, etc. Thus, in particular embodiments, the bioactivity ofthe conjugate is higher when administered to the mammal at the higher ofthe two glucose concentrations (e.g., 300 vs. 100 mg/dL glucose). Inparticular embodiments, the bioactivity of the conjugate is at least 25%(e.g., at least 50% or at least 100%) higher when administered to themammal at the higher of the two glucose concentrations (e.g., 300 vs.100 mg/dL glucose).

In particular embodiments, the conjugate includes an insulin molecule asthe drug. According to such embodiments, the PD behavior for insulin canbe observed by comparing the time to reach minimum blood glucoseconcentration (T_(nadir)), the duration over which the blood glucoselevel remains below a particular percentage of the initial value (e.g.,70% of initial value or T_(70% BGL)), etc.

In general, it will be appreciated that any of the PK and PDcharacteristics discussed in this section can be determined according toany of a variety of published pharmacokinetic and pharmacodynamicmethods (e.g., see Baudys et al., Bioconjugate Chem. 9:176-183, 1998 formethods suitable for subcutaneous delivery). It is also to be understoodthat the PK and/or PD properties may be measured in any mammal (e.g., ahuman, a rat, a cat, a minipig, a dog, etc.). In particular embodiments,PK and/or PD properties are measured in a human. In particularembodiments, PK and/or PD properties are measured in a rat. Inparticular embodiments, PK and/or PD properties are measured in aminipig. In particular embodiments, PK and/or PD properties are measuredin a dog.

It will also be appreciated that while the foregoing was described inthe context of glucose-responsive conjugates, the same properties andassays apply to conjugates that are responsive to other saccharidesincluding exogenous saccharides, e.g., mannose, L-fucose, N-acetylglucosamine, alpha-methyl mannose, etc. As discussed in more detailbelow and in the Examples, instead of comparing PK and/or PD propertiesunder fasted and hyperglycemic conditions, the PK and/or PD propertiesmay be compared under fasted conditions with and without administrationof the exogenous saccharide. It is to be understood that conjugates canbe designed that respond to different C_(max) values of a givenexogenous saccharide.

Ligand(s)

In general, the insulin conjugates comprise an insulin or insulin analogmolecule covalently attached to at least one bi-dentate linker havingtwo ligands wherein at least one of the ligands (the first ligand)comprises or consists of a saccharide, which is fucose, and the otherligand (the second ligand) comprises or consists of one or moresaccharides. In particular embodiments, the insulin conjugates mayfurther include one or more linear linkers, each comprising a singleligand, which comprises or consist of one or more saccharides. Inparticular embodiments, the insulin conjugates may further include oneor more branched linkers that each includes at least two, three, four,five, or more ligands, where each ligand independently comprises orconsists of one or more saccharides. When more than one ligand ispresent the ligands may have the same or different chemical structures.

In particular embodiments, the ligands are capable of competing with asaccharide (e.g., glucose, alpha-methylmannose, or mannose) for bindingto an endogenous saccharide-binding molecule (e.g., without limitationsurfactant proteins A and D or members of the selectin family). Inparticular embodiments, the ligands are capable of competing with asaccharide (e.g., glucose, alpha-methylmannose, or mannose) for bindingto cell-surface sugar receptor (e.g., without limitation macrophagemannose receptor, glucose transporter ligands, endothelial cell sugarreceptors, or hepatocyte sugar receptors). In particular embodiments,the ligands are capable of competing with glucose for binding to anendogenous glucose-binding molecule (e.g., without limitation surfactantproteins A and D or members of the selectin family). In particularembodiments, the ligands are capable of competing with glucose oralpha-mewthylmannose for binding to the human macrophage mannosereceptor 1 (MRC1). In particular embodiments, the ligands are capable ofcompeting with a saccharide for binding to a non-human lectin (e.g., ConA). In particular embodiments, the ligands are capable of competing withglucose, alpha-methylmannose, or mannose for binding to a non-humanlectin (e.g., Con A). Exemplary glucose-binding lectins includecalnexin, calreticulin, N-acetylglucosamine receptor, selectin,asialoglycoprotein receptor, collectin (mannose-binding lectin), mannosereceptor, aggrecan, versican, pisum sativum agglutinin (PSA), vicia fabalectin, lens culinaris lectin, soybean lectin, peanut lectin, lathyrusochrus lectin, sainfoin lectin, sophora japonica lectin, bowringiamilbraedii lectin, concanavalin A (Con A), and pokeweed mitogen.

In particular embodiments, the ligand(s) other than the first ligandcomprising or consisting of the saccharide fucose may have the samechemical structure as glucose or may be a chemically related species ofglucose, e.g., glucosamine. In various embodiments, it may beadvantageous for the ligand(s) to have a different chemical structurefrom glucose, e.g., in order to fine tune the glucose response of theconjugate. For example, in particular embodiments, one might use aligand that includes glucose, mannose, L-fucose or derivatives of these(e.g., alpha-L-fucopyranoside, mannosamine, beta-linked N-acetylmannosamine, methylglucose, methylmannose, ethylglucose, ethylmannose,propylglucose, propylmannose, etc.) and/or higher order combinations ofthese (e.g., a bimannose, linear and/or branched trimannose, etc.).

In particular embodiments, the ligand(s) include(s) a monosaccharide. Inparticular embodiments, the ligand(s) include(s) a disaccharide. Inparticular embodiments, the ligand(s) include(s) a trisaccharide. Insome embodiments, the ligand(s) comprise a saccharide and one or moreamine groups. In some embodiments, the ligand(s) comprise a saccharideand ethyl group. In particular embodiments, the saccharide and aminegroup are separated by a C₁-C₆ alkyl group, e.g., a C₁-C₃ alkyl group.In some embodiments, the ligand is aminoethylglucose (AEG). In someembodiments, the ligand is aminoethylmannose (AEM). In some embodiments,the ligand is aminoethylbimannose (AEBM). In some embodiments, theligand is aminoethyltrimannose (AETM). In some embodiments, the ligandis β-aminoethyl-N-acetylglucosamine (AEGA). In some embodiments, theligand is aminoethylfucose (AEF). In particular embodiments, thesaccharide is of the “D” configuration and in other embodiments, thesaccharide is of the “L” configuration. Below are the structures ofexemplary saccharides having an amine group separated from thesaccharide by a C₂ ethyl group wherein R may be hydrogen or a carbonylgroup of the linker. Other exemplary ligands will be recognized by thoseskilled in the art.

Insulin

As used herein, the term “insulin” or “insulin molecule” encompasses allsalt and non-salt forms of the insulin molecule. It will be appreciatedthat the salt form may be anionic or cationic depending on the insulinmolecule. By “insulin” or “an insulin molecule” we intend to encompassboth wild-type insulin and modified forms of insulin as long as they arebioactive (i.e., capable of causing a detectable reduction in glucosewhen administered in vivo). Wild-type insulin includes insulin from anyspecies whether in purified, synthetic or recombinant form (e.g., humaninsulin, porcine insulin, bovine insulin, rabbit insulin, sheep insulin,etc.). A number of these are available commercially, e.g., fromSigma-Aldrich (St. Louis, Mo.). A variety of modified forms of insulinare known in the art (e.g. see Crotty and Reynolds, Pediatr. Emerg.Care. 23:903-905, 2007 and Gerich, Am. J. Med. 113:308-16, 2002 andreferences cited therein). Modified forms of insulin (insulin analogs)may be chemically modified (e.g., by addition of a chemical moiety suchas a PEG group or a fatty acyl chain as described below) and/or mutated(i.e., by addition, deletion or substitution of one or more aminoacids).

In particular embodiments, an insulin molecule of the present disclosurewill differ from a wild-type insulin by 1-10 (e.g., 1-9, 1-8, 1-7, 1-6,1-5, 1-4, 1-3, 1-2, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-9, 3-8, 3-7,3-6, 3-5, 3-4, 4-9, 4-8, 4-7, 4-6, 4-5, 5-9, 5-8, 5-7, 5-6, 6-9, 6-8,6-7, 7-9, 7-8, 8-9, 9, 8, 7, 6, 5, 4, 3, 2 or 1) amino acidsubstitutions, additions and/or deletions.

In particular embodiments, an insulin molecule of the present disclosurewill differ from a wild-type insulin by amino acid substitutions only.In particular embodiments, an insulin molecule of the present disclosurewill differ from a wild-type insulin by amino acid additions only. Inparticular embodiments, an insulin molecule of the present disclosurewill differ from wild-type insulin by both amino acid substitutions andadditions. In particular embodiments, an insulin molecule of the presentdisclosure will differ from a wild-type insulin by both amino acidsubstitutions and deletions.

In particular embodiments, amino acid substitutions may be made on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.In particular embodiments, a substitution may be conservative, that is,one amino acid is replaced with one of similar shape and charge.Conservative substitutions are well known in the art and typicallyinclude substitutions within the following groups: glycine, alanine;valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and tyrosine,phenylalanine. In particular embodiments, the hydrophobic index of aminoacids may be considered in choosing suitable mutations. The importanceof the hydrophobic amino acid index in conferring interactive biologicalfunction on a polypeptide is generally understood in the art.Alternatively, the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity. The importance ofhydrophilicity in conferring interactive biological function of apolypeptide is generally understood in the art. The use of thehydrophobic index or hydrophilicity in designing polypeptides is furtherdiscussed in U.S. Pat. No. 5,691,198.

The wild-type sequence of human insulin (A-chain and B-chain) is shownbelow.

A-Chain (SEQ ID NO: 1): GIVEQCCTSICSLYQLENYCN B-Chain (SEQ ID NO: 2):FVNQHLCGSHLVEALYLVCGERGFFYTPKT

In various embodiments, an insulin molecule of the present disclosure ismutated at the B28 and/or B29 positions of the B-peptide sequence. Forexample, insulin lispro (HUMALOG®) is a rapid acting insulin mutant inwhich the penultimate lysine and proline residues on the C-terminal endof the B-peptide have been reversed (Lys^(B28)Pro^(B29)-human insulin)(SEQ ID NO:3). This modification blocks the formation of insulinmultimers. Insulin aspart (NOVOLOG®) is another rapid acting insulinmutant in which proline at position B28 has been substituted withaspartic acid (Asp^(B28)-human insulin) (SEQ ID NO:4). This mutant alsoprevents the formation of multimers. In some embodiments, mutation atpositions B28 and/or B29 is accompanied by one or more mutationselsewhere in the insulin polypeptide. For example, insulin glulisine(APIDRA®) is yet another rapid acting insulin mutant in which asparticacid at position B3 has been replaced by a lysine residue and lysine atposition B29 has been replaced with a glutamic acid residue(Lys^(B3)Glu^(B29)-human insulin) (SEQ ID NO:5).

In various embodiments, an insulin molecule of the present disclosurehas an isoelectric point that is shifted relative to human insulin. Insome embodiments, the shift in isoelectric point is achieved by addingone or more arginine residues to the N-terminus of the insulin A-peptideand/or the C-terminus of the insulin B-peptide. Examples of such insulinpolypeptides include Arg^(A0)-human insulin, Arg^(B31)Arg^(B32)-humaninsulin, Gly^(A21)Arg^(B31)Arg^(B32)-human insulin,Arg^(A0)Arg^(B31)Arg^(B32)-human insulin, andArg^(A0)Gly^(A21)Arg^(B31)Arg^(B32)-human insulin. By way of furtherexample, insulin glargine (LANTUS®) is an exemplary long acting insulinmutant in which Asp^(A21) has been replaced by glycine (SEQ ID NO:6),and two arginine residues have been added to the C-terminus of theB-peptide (SEQ ID NO:7). The effect of these changes is to shift theisoelectric point, producing a solution that is completely soluble at pH4. Thus, in some embodiments, an insulin molecule of the presentdisclosure comprises an A-peptide sequence wherein A21 is Gly andB-peptide sequence wherein B31 and B32 are Arg-Arg. It is to beunderstood that the present disclosure encompasses all single andmultiple combinations of these mutations and any other mutations thatare described herein (e.g., Gly^(A21)-human insulin,Gly^(A21)Arg^(B31)-human insulin, Arg^(B31)Arg^(B32)-human insulin,Arg^(B31)-human insulin).

In various embodiments, an insulin molecule of the present disclosure istruncated. For example, in particular embodiments, a B-peptide sequenceof an insulin polypeptide of the present disclosure is missing B1, B2,B3, B26, B27, B28, B29 and/or B30. In particular embodiments,combinations of residues are missing from the B-peptide sequence of aninsulin polypeptide of the present disclosure. For example, theB-peptide sequence may be missing residues B(1-2), B(1-3), B(29-30),B(28-30), B(27-30) and/or B(26-30). In some embodiments, these deletionsand/or truncations apply to any of the aforementioned insulin molecules(e.g., without limitation to produce des(B30)-insulin lispro,des(B30)-insulin aspart, des(B30)-insulin glulisine, des(B30)-insulinglargine, etc.).

In some embodiments, an insulin molecule contains additional amino acidresidues on the N- or C-terminus of the A or B-peptide sequences. Insome embodiments, one or more amino acid residues are located atpositions A0, A21, B0 and/or B31. In some embodiments, one or more aminoacid residues are located at position A0. In some embodiments, one ormore amino acid residues are located at position A21. In someembodiments, one or more amino acid residues are located at position B0.In some embodiments, one or more amino acid residues are located atposition B31. In particular embodiments, an insulin molecule does notinclude any additional amino acid residues at positions A0, A21, B0 orB31.

In particular embodiments, an insulin molecule of the present disclosureis mutated such that one or more amidated amino acids are replaced withacidic forms. For example, asparagine may be replaced with aspartic acidor glutamic acid. Likewise, glutamine may be replaced with aspartic acidor glutamic acid. In particular, Asn^(A18), Asn^(A21), or Asn^(B3), orany combination of those residues, may be replaced by aspartic acid orglutamic acid. Gln^(A15) or Gln^(B4), or both, may be replaced byaspartic acid or glutamic acid. In particular embodiments, an insulinmolecule has aspartic acid at position A21 or aspartic acid at positionB3, or both.

One skilled in the art will recognize that it is possible to mutate yetother amino acids in the insulin molecule while retaining biologicalactivity. For example, without limitation, the following modificationsare also widely accepted in the art: replacement of the histidineresidue of position B10 with aspartic acid (His^(B10)→Asp^(B10));replacement of the phenylalanine residue at position B1 with asparticacid (Phe^(B1)→Asp^(B1)); replacement of the threonine residue atposition B30 with alanine (Thr^(B30)→Ala^(B30)); replacement of thetyrosine residue at position B26 with alanine (Tyr^(B26)→Ala^(B26)); andreplacement of the serine residue at position B9 with aspartic acid(Ser^(B9)→Asp^(B9)).

In various embodiments, an insulin molecule of the present disclosurehas a protracted profile of action. Thus, in particular embodiments, aninsulin molecule of the present disclosure may be acylated with a fattyacid. That is, an amide bond is formed between an amino group on theinsulin molecule and the carboxylic acid group of the fatty acid. Theamino group may be the alpha-amino group of an N-terminal amino acid ofthe insulin molecule, or may be the epsilon-amino group of a lysineresidue of the insulin molecule. An insulin molecule of the presentdisclosure may be acylated at one or more of the three amino groups thatare present in wild-type human insulin or may be acylated on lysineresidue that has been introduced into the wild-type human insulinsequence. In particular embodiments, an insulin molecule may be acylatedat position B1. In particular embodiments, an insulin molecule may beacylated at position B29. In particular embodiments, the fatty acid isselected from myristic acid (C14), pentadecylic acid (C15), palmiticacid (C16), heptadecylic acid (C17) and stearic acid (C18). For example,insulin detemir (LEVEMIR®) is a long acting insulin mutant in whichThr^(B30) has been deleted, and a C14 fatty acid chain (myristic acid)has been attached to Lys^(B29).

In some embodiments, the N-terminus of the A-peptide, the N-terminus ofthe B-peptide, the epsilon-amino group of Lys at position B29 or anyother available amino group in an insulin molecule of the presentdisclosure is covalently linked to a fatty acid moiety of generalformula:

wherein R^(F) is hydrogen or a C₁₋₃₀ alkyl group. In some embodiments,R^(F) is a C₁₋₂₀ alkyl group, a C₃₋₁₉ alkyl group, a C₅₋₁₈ alkyl group,a C₆₋₁₇ alkyl group, a C₈₋₁₆ alkyl group, a C₁₀₋₁₅ alkyl group, or aC₁₂₋₁₄ alkyl group. In particular embodiments, the insulin polypeptideis conjugated to the moiety at the A1 position. In particularembodiments, the insulin polypeptide is conjugated to the moiety at theB1 position. In particular embodiments, the insulin polypeptide isconjugated to the moiety at the epsilon-amino group of Lys at positionB29. In particular embodiments, position B28 of the insulin molecule isLys and the epsilon-amino group of Lys^(B28) is conjugated to the fattyacid moiety. In particular embodiments, position B3 of the insulinmolecule is Lys and the epsilon-amino group of Lys^(B3) is conjugated tothe fatty acid moiety. In some embodiments, the fatty acid chain is 8-20carbons long. In some embodiments, the fatty acid is octanoic acid (C8),nonanoic acid (C9), decanoic acid (C10), undecanoic acid (C11),dodecanoic acid (C12), or tridecanoic acid (C13). In particularembodiments, the fatty acid is myristic acid (C14), pentadecanoic acid(C15), palmitic acid (C16), heptadecanoic acid (C17), stearic acid(C18), nonadecanoic acid (C19), or arachidic acid (C20).

In various embodiments, an insulin molecule of the present disclosureincludes the three wild-type disulfide bridges (i.e., one betweenposition 7 of the A-chain and position 7 of the B-chain, a secondbetween position 20 of the A-chain and position 19 of the B-chain, and athird between positions 6 and 11 of the A-chain). In particularembodiments, an insulin molecule is mutated such that the site ofmutation is used as a conjugation point, and conjugation at the mutatedsite reduces binding to the insulin receptor (e.g., Lys^(A3)). Inparticular other embodiments, conjugation at an existing wild-type aminoacid or terminus reduces binding to the insulin receptor (e.g.,Gly^(A1)). In some embodiments, an insulin molecule is conjugated atposition A4, A5, A8, A9, or B30. In particular embodiments, theconjugation at position A4, A5, A8, A9, or B30 takes place via awild-type amino acid side chain (e.g., Glu^(A4)). In particular otherembodiments, an insulin molecule is mutated at position A4, A5, A8, A9,or B30 to provide a site for conjugation (e.g., Lys^(A4), Lys^(A5),Lys^(A8), Lys^(A9), or Lys^(B30)).

Methods for conjugating insulin molecules are described below. Inparticular embodiments an insulin molecule is conjugated to a linker viathe A1 amino acid residue. In particular embodiments the A1 amino acidresidue is glycine. It is to be understood however, that the presentdisclosure is not limited to N-terminal conjugation and that inparticular embodiments an insulin molecule may be conjugated via anon-terminal A-chain amino acid residue. In particular, the presentdisclosure encompasses conjugation via the epsilon-amine group of alysine residue present at any position in the A-chain (wild-type orintroduced by site-directed mutagenesis). It will be appreciated thatdifferent conjugation positions on the A-chain may lead to differentreductions in insulin activity. In particular embodiments, an insulinmolecule is conjugated to the linker via the B1 amino acid residue. Inparticular embodiments the B1 amino acid residue is phenylalanine. It isto be understood however, that the present disclosure is not limited toN-terminal conjugation and that in particular embodiments an insulinmolecule may be conjugated via a non-terminal B-chain amino acidresidue. In particular, the present disclosure encompasses conjugationvia the epsilon-amine group of a lysine residue present at any positionin the B-chain (wild-type or introduced by site-directed mutagenesis).For example, in particular embodiments an insulin molecule may beconjugated via the B29 lysine residue. In the case of insulin glulisine,conjugation to the at least one ligand via the B3 lysine residue may beemployed. It will be appreciated that different conjugation positions onthe B-chain may lead to different reductions in insulin activity.

In particular embodiments, the ligands are conjugated to more than oneconjugation point on the insulin molecule. For example, an insulinmolecule can be conjugated at both the A1 N-terminus and the B29 lysine.In some embodiments, amide conjugation takes place in carbonate bufferto conjugate at the B29 and A1 positions, but not at the B1 position. Inother embodiments, an insulin molecule can be conjugated at the A1N-terminus, the B1 N-terminus, and the B29 lysine. In yet otherembodiments, protecting groups are used such that conjugation takesplace at the B1 and B29 or B1 and A1 positions. It will be appreciatedthat any combination of conjugation points on an insulin molecule may beemployed. In some embodiments, at least one of the conjugation points isa mutated lysine residue, e.g., Lys^(A3).

Exemplary Insulin Conjugates

In various embodiments, the insulin conjugate of the present disclosurecomprises an insulin or insulin analog molecule conjugated to at leastone bi-dentate linker wherein at least one arm of the bi-dentate linkeris attached to the ligand aminoethylfucose (AEF). The other arm of thebi-dentate linker may be conjugated to the ligand AEF and/or one or moreligands that are independently selected from the group consisting ofaminoethylglucose (AEG), aminoethylmannose (AEM), aminoethylbimannose(AEBM), aminoethyltrimannose (AETM), p-aminoethyl-N-acetylglucosamine(AEGA), and aminoethylfucose (AEF). In particular embodiments, theinsulin molecule is conjugated via the A1 amino acid residue. Inparticular embodiments, the insulin molecule is conjugated via the B1amino acid residue. In particular embodiments, the insulin molecule isconjugated via the epsilon-amino group of Lys^(B29). In particularembodiments, the insulin molecule is an analog that comprises a lysineat position B28 (Lys^(B28)) and the insulin molecule is conjugated viathe epsilon-amino group of Lys^(B28), for example, insulin lisproconjugated via the epsilon-amino group of Lys^(B28). In particularembodiments, the insulin molecule is an analog that comprises a lysineat position B3 (Lys^(B3)) and the insulin molecule is conjugated via theepsilon-amino group of Lys^(B3), for example, insulin glulisineconjugated via the epsilon-amino group of Lys^(B3).

In particular embodiments, the insulin or insulin molecule of the aboveinsulin conjugate may be conjugated to one or more additional linkersattached to one or more ligands, each ligand independently selected fromaminoethylglucose (AEG), aminoethylmannose (AEM), aminoethylbimannose(AEBM) ligands, aminoethyltrimannose (AETM) ligands,β-aminoethyl-N-acetylglucosamine (AEGA), and aminoethylfucose (AEF). Theadditional linkers may be linear, bi-dentate, tri-dentate,quadri-dentate, etc. wherein each arm of the linker comprises a ligandwhich may indenpendently be selected from aminoethylglucose (AEG),aminoethylmannose (AEM), aminoethylbimannose (AEBM) ligands,aminoethyltrimannose (AETM) ligands, β-aminoethyl-N-acetylglucosamine(AEGA), and aminoethylfucose (AEF).

Thus, in particular embodiments, the insulin conjugate may comprise orconsist of a bi-dentate linker wherein at least one arm of thebi-dentate linker is attached to the ligand aminoethylfucose (AEF)conjugated to the amino group at position A1 of the insulin or insulinanalog; or the amino group at position B1 of the insulin or insulinanalog; or the amino group at position B3 of the insulin analog; or theamino group at position B28 of the insulin analog; or the amino group atposition B29 of the insulin or insulin analog.

In particular embodiments, the insulin conjugate may comprise or consistof two bi-dentate linkers wherein a first bi-dentate linker, which hasthe ligand aminoethylfucose (AEF) attached to one arm of the firstbi-dentate linker and a ligand selected from aminoethylglucose (AEG),aminoethylmannose (AEM), aminoethylbimannose (AEBM),aminoethyltrimannose (AETM), β-aminoethyl-N-acetylglucosamine (AEGA),and aminoethylfucose (AEF) attached to the other arm of the bi-dentatelinker, is conjugated to the amino group at position A1 and a secondbi-dentate linker attached to one or more ligands, each ligandindependently selected from aminoethylglucose (AEG), aminoethylmannose(AEM), aminoethylbimannose (AEBM), aminoethyltrimannose (AETM),β-aminoethyl-N-acetylglucosamine (AEGA), and aminoethylfucose (AEF) isconjugated to the amino group at position B1, B3, B28, or B29.

In particular embodiments, the insulin conjugate may comprise or consistof two bi-dentate linkers wherein a first bi-dentate linker, which hasthe ligand aminoethylfucose (AEF) attached to one arm of the firstbi-dentate linker and a ligand selected from aminoethylglucose (AEG),aminoethylmannose (AEM), aminoethylbimannose (AEBM),aminoethyltrimannose (AETM), β-aminoethyl-N-acetylglucosamine (AEGA),and aminoethylfucose (AEF) attached to the other arm of the bi-dentatelinker, is conjugated to the amino group at position B1 and a secondbi-dentate linker attached to one or more ligands, each ligandindependently selected from aminoethylglucose (AEG), aminoethylmannose(AEM), aminoethylbimannose (AEBM), aminoethyltrimannose (AETM),β-aminoethyl-N-acetylglucosamine (AEGA), and aminoethylfucose (AEF) isconjugated to the amino group at position A1, B3, B28, or B29.

In particular embodiments, the insulin conjugate may comprise or consistof two bi-dentate linkers wherein a first bi-dentate linker, which hasthe ligand aminoethylfucose (AEF) attached to one arm of the firstbi-dentate linker and a ligand selected from aminoethylglucose (AEG),aminoethylmannose (AEM), aminoethylbimannose (AEBM),aminoethyltrimannose (AETM), β-aminoethyl-N-acetylglucosamine (AEGA),and aminoethylfucose (AEF) attached to the other arm of the bi-dentatelinker, is conjugated to the amino group at position B3 and a secondbi-dentate linker attached to one or more ligands, each ligandindependently selected from aminoethylglucose (AEG), aminoethylmannose(AEM), aminoethylbimannose (AEBM), aminoethyltrimannose (AETM),β-aminoethyl-N-acetylglucosamine (AEGA), and aminoethylfucose (AEF) isconjugated to the amino group at position B1, A1, B28, or B29.

In particular embodiments, the insulin conjugate may comprise or consistof two bi-dentate linkers wherein a first bi-dentate linker, which hasthe ligand aminoethylfucose (AEF) attached to one arm of the firstbi-dentate linker and a ligand selected from aminoethylglucose (AEG),aminoethylmannose (AEM), aminoethylbimannose (AEBM),aminoethyltrimannose (AETM), β-aminoethyl-N-acetylglucosamine (AEGA),and aminoethylfucose (AEF) attached to the other arm of the bi-dentatelinker, is conjugated to the amino group at position B28 and a secondbi-dentate linker attached to one or more ligands, each ligandindependently selected from aminoethylglucose (AEG), aminoethylmannose(AEM), aminoethylbimannose (AEBM), aminoethyltrimannose (AETM),β-aminoethyl-N-acetylglucosamine (AEGA), and aminoethylfucose (AEF) isconjugated to the amino group at position B1, B3, A1, or B29.

In particular embodiments, the insulin conjugate may comprise or consistof two bi-dentate linkers wherein a first bi-dentate linker, which hasthe ligand aminoethylfucose (AEF) attached to one arm of the firstbi-dentate linker and a ligand selected from aminoethylglucose (AEG),aminoethylmannose (AEM), aminoethylbimannose (AEBM),aminoethyltrimannose (AETM), β-aminoethyl-N-acetylglucosamine (AEGA),and aminoethylfucose (AEF) attached to the other arm of the bi-dentatelinker, is conjugated to the amino group at position B29 and a secondbi-dentate linker attached to one or more ligands, each ligandindependently selected from aminoethylglucose (AEG), aminoethylmannose(AEM), aminoethylbimannose (AEBM), aminoethyltrimannose (AETM),β-aminoethyl-N-acetylglucosamine (AEGA), and aminoethylfucose (AEF) isconjugated to the amino group at position B1, B3, B28, or A1.

In particular embodiments, the insulin conjugate may comprise or consistof three bi-dentate linkers wherein a first bi-dentate linker, which hasthe ligand aminoethylfucose (AEF) attached to one arm of the firstbi-dentate linker and a ligand selected from aminoethylglucose (AEG),aminoethylmannose (AEM), aminoethylbimannose (AEBM),aminoethyltrimannose (AETM), β-aminoethyl-N-acetylglucosamine (AEGA),and aminoethylfucose (AEF) attached to the other arm of the bi-dentatelinker, is conjugated to the amino group at position A1; a secondbi-dentate linker attached to one or more ligands, each ligandindependently selected from aminoethylglucose (AEG), aminoethylmannose(AEM), aminoethylbimannose (AEBM), aminoethyltrimannose (AETM),β-aminoethyl-N-acetylglucosamine (AEGA), and aminoethylfucose (AEF) isconjugated to the amino group at position B1; and, a third bi-dentatelinker attached to one or more ligands, each ligand independentlyselected from aminoethylglucose (AEG), aminoethylmannose (AEM),aminoethylbimannose (AEBM), aminoethyltrimannose (AETM),β-aminoethyl-N-acetylglucosamine (AEGA), and aminoethylfucose (AEF) isconjugated to the amino group at position B3, B28, or B29.

In particular embodiments, the insulin conjugate may comprise or consistof four bi-dentate linkers wherein a first bi-dentate linker, which hasthe ligand aminoethylfucose (AEF) attached to one arm of the firstbi-dentate linker and a ligand selected from aminoethylglucose (AEG),aminoethylmannose (AEM), aminoethylbimannose (AEBM),aminoethyltrimannose (AETM), β-aminoethyl-N-acetylglucosamine (AEGA),and aminoethylfucose (AEF) attached to the other arm of the bi-dentatelinker, is conjugated to the amino group at position A1; a secondbi-dentate linker attached to one or more ligands, each ligandindependently selected from aminoethylglucose (AEG), aminoethylmannose(AEM), aminoethylbimannose (AEBM), aminoethyltrimannose (AETM),β-aminoethyl-N-acetylglucosamine (AEGA), and aminoethylfucose (AEF) isconjugated to the amino group at position B1; a third bi-dentate linkerattached to one or more ligands, each ligand independently selected fromaminoethylglucose (AEG), aminoethylmannose (AEM), aminoethylbimannose(AEBM), aminoethyltrimannose (AETM), β-aminoethyl-N-acetylglucosamine(AEGA), and aminoethylfucose (AEF) is conjugated to the amino group atposition B3; and a fourth bi-dentate linker attached to one or moreligands, each ligand independently selected from aminoethylglucose(AEG), aminoethylmannose (AEM), aminoethylbimannose (AEBM),aminoethyltrimannose (AETM), β-aminoethyl-N-acetylglucosamine (AEGA),and aminoethylfucose (AEF) is conjugated to the amino group at positionB28 or B29.

In particular embodiments, the insulin conjugate may comprise or consistof (a) a bi-dentate linker wherein at least one arm of the bi-dentatelinker is attached to the ligand aminoethylfucose (AEF) conjugated tothe amino group at position A1; or the amino group at position B1; orthe amino group at position B3; or the amino group at position B28; orthe amino group at position B29 and (b) a linear or tri-dentate linkerattached to one or more ligands, each ligand independently selected fromaminoethylglucose (AEG), aminoethylmannose (AEM), aminoethylbimannose(AEBM), aminoethyltrimannose (AETM), β-aminoethyl-N-acetylglucosamine(AEGA), and aminoethylfucose (AEF) conjugated to the amino group atposition A1; or the amino group at position B1; or the amino group atposition B3; or the amino group at position B28; or the amino group atposition B29, whichever position is not occupied by the bi-dentatelinker.

In particular embodiments, the insulin conjugate may comprise or consistof (a) a bi-dentate linker, which has the ligand aminoethylfucose (AEF)attached to one arm of the first bi-dentate linker and a ligand selectedfrom aminoethylglucose (AEG), aminoethylmannose (AEM),aminoethylbimannose (AEBM), aminoethyltrimannose (AETM),β-aminoethyl-N-acetylglucosamine (AEGA), and aminoethylfucose (AEF)attached to the other arm of the bi-dentate linker, conjugated to theamino group at position A1 and (b) a linear or tri-dentate linkerattached to one or more ligands, each ligand independently selected fromaminoethylglucose (AEG), aminoethylmannose (AEM), aminoethylbimannose(AEBM), aminoethyltrimannose (AETM), β-aminoethyl-N-acetylglucosamine(AEGA), and aminoethylfucose (AEF) conjugated to the amino group atposition B1, B3, B28, or B29.

In particular embodiments, the insulin conjugate may comprise or consistof (a) a bi-dentate linker, which has the ligand aminoethylfucose (AEF)attached to one arm of the first bi-dentate linker and a ligand selectedfrom aminoethylglucose (AEG), aminoethylmannose (AEM),aminoethylbimannose (AEBM), aminoethyltrimannose (AETM),β-aminoethyl-N-acetylglucosamine (AEGA), and aminoethylfucose (AEF)attached to the other arm of the bi-dentate linker, conjugated to theamino group at position B1 and (b) a linear or tri-dentate linkerattached to one or more ligands, each ligand independently selected fromaminoethylglucose (AEG), aminoethylmannose (AEM), aminoethylbimannose(AEBM), aminoethyltrimannose (AETM), β-aminoethyl-N-acetylglucosamine(AEGA), and aminoethylfucose (AEF) conjugated to the amino group atposition A1, B3, B28, or B29.

In particular embodiments, the insulin conjugate may comprise or consistof (a) a bi-dentate linker, which has the ligand aminoethylfucose (AEF)attached to one arm of the first bi-dentate linker and a ligand selectedfrom aminoethylglucose (AEG), aminoethylmannose (AEM),aminoethylbimannose (AEBM), aminoethyltrimannose (AETM),β-aminoethyl-N-acetylglucosamine (AEGA), and aminoethylfucose (AEF)attached to the other arm of the bi-dentate linker, conjugated to theamino group at position B3 and (b) a linear or tri-dentate linkerattached to one or more ligands, each ligand independently selected fromaminoethylglucose (AEG), aminoethylmannose (AEM), aminoethylbimannose(AEBM), aminoethyltrimannose (AETM), β-aminoethyl-N-acetylglucosamine(AEGA), and aminoethylfucose (AEF) conjugated to the amino group atposition B1, A1, B28, or B29.

In particular embodiments, the insulin conjugate may comprise or consistof (a) a bi-dentate linker, which has the ligand aminoethylfucose (AEF)attached to one arm of the first bi-dentate linker and a ligand selectedfrom aminoethylglucose (AEG), aminoethylmannose (AEM),aminoethylbimannose (AEBM), aminoethyltrimannose (AETM),β-aminoethyl-N-acetylglucosamine (AEGA), and aminoethylfucose (AEF)attached to the other arm of the bi-dentate linker, conjugated to theamino group at position B28 and (b) a linear or tri-dentate linkerattached to one or more ligands, each ligand independently selected fromaminoethylglucose (AEG), aminoethylmannose (AEM), aminoethylbimannose(AEBM), aminoethyltrimannose (AETM), β-aminoethyl-N-acetylglucosamine(AEGA), and aminoethylfucose (AEF) conjugated to the amino group atposition B1, B3, A1, or B29.

In particular embodiments, the insulin conjugate may comprise or consistof (a) a bi-dentate linker, which has the ligand aminoethylfucose (AEF)attached to one arm of the first bi-dentate linker and a ligand selectedfrom aminoethylglucose (AEG), aminoethylmannose (AEM),aminoethylbimannose (AEBM), aminoethyltrimannose (AETM),β-aminoethyl-N-acetylglucosamine (AEGA), and aminoethylfucose (AEF)attached to the other arm of the bi-dentate linker, conjugated to theamino group at position B29 and (b) a linear or tri-dentate linkerattached to one or more ligands, each ligand independently selected fromaminoethylglucose (AEG), aminoethylmannose (AEM), aminoethylbimannose(AEBM), aminoethyltrimannose (AETM), β-aminoethyl-N-acetylglucosamine(AEGA), and aminoethylfucose (AEF) conjugated to the amino group atposition B1, B3, B28, or A1.

In particular embodiments, the insulin conjugate may comprise or consistof (a) a bi-dentate linker, which has the ligand aminoethylfucose (AEF)attached to one arm of the first bi-dentate linker and a ligand selectedfrom aminoethylglucose (AEG), aminoethylmannose (AEM),aminoethylbimannose (AEBM), aminoethyltrimannose (AETM),β-aminoethyl-N-acetylglucosamine (AEGA), and aminoethylfucose (AEF)attached to the other arm of the bi-dentate linker; (b) a first linearor tri-dentate linker attached to one or more ligands, each ligandindependently selected from aminoethylglucose (AEG), aminoethylmannose(AEM), aminoethylbimannose (AEBM), aminoethyltrimannose (AETM),β-aminoethyl-N-acetylglucosamine (AEGA), and aminoethylfucose (AEF); and(c) a second linear or tri-dentate linker attached to one or moreligands, each ligand independently selected from aminoethylglucose(AEG), aminoethylmannose (AEM), aminoethylbimannose (AEBM),aminoethyltrimannose (AETM), β-aminoethyl-N-acetylglucosamine (AEGA),and aminoethylfucose (AEF), wherein each linker is each conjugated to aamino group at position A1,B1, B3, B28, or B29 with the proviso thateach occupies a separate position such that three sites in total areoccupied.

Insulin Conjugates

This section describes some exemplary insulin or insulin analogconjugates.

In particular embodiments, provided are insulin and insulin analogconjugates comprising at least one fucose wherein the conjugate ischaracterized as having a ratio of EC₅₀ or IP as determined by afunctional insulin receptor phosphorylation assay verses the IC₅₀ or IPas determined by a competition binding assay at the macrophage mannosereceptor is about 0.5:1 to about 1:100; about 1:1 to about 1:50; about1:1 to about 1:20; or about 1:1 to about 1:10. In further aspects, theabove conjugate comprises an insulin or insulin analog moleculecovalently attached to at least one branched linker having a first armand second arm, wherein the first arm is linked to a first ligand thatincludes a first saccharide and the second arm is linked to a secondligand that includes a second saccharide and wherein the firstsaccharide is fucose and wherein the conjugate is characterized ashaving a ratio of EC₅₀ or IP as determined by a functional insulinreceptor phosphorylation assay verses the IC₅₀ or IP as determined by acompetition binding assay at the macrophage mannose receptor is about0.5:1 to about 1:100; about 1:1 to about 1:50; about 1:1 to about 1:20;or about 1:1 to about 1:10. In particular aspects, the second saccharideis a fucose, mannose, glucosamine, glucose, bisaccharide, trisaccharide,tetrasaccharide, branched trisaccharide, bimannose, trimannose,tetramannose, or branched trimannose.

The term “IP” refers to the inflection point, which is a point on acurve at which the curvature or concavity changes sign from plus tominus or from minus to plus. In general, IP is usually equivalent to theEC₅₀ or IC₅₀.

In particular aspects, the IC₅₀ or IP as determined by a competitionbinding assay at the macrophage mannose receptor may be less than about100 nM and greater than about 0.5 nM. In particular aspects, the IC₅₀ orIP is less than about 50 nM and greater than about 1 nM; less than about25 nM and greater than about 1 nM; or less than about 20 nM and greaterthan about 1 nM. In particular aspects, the IC₅₀ or IP as determined bya functional insulin receptor phosphorylation assay may be less thanabout 100 nM and greater than about 0.5 nM. In particular aspects, theIC₅₀ or IP is less than about 50 nM and greater than about 1 nM; lessthan about 25 nM and greater than about 1 nM; or less than about 20 nMand greater than about 1 nM.

In various embodiments, the conjugates may have the general formula (I):

wherein:

-   each occurrence of

represents a potential repeat within a branch of the conjugate;

-   each occurrence of    is independently a covalent bond, a carbon atom, a heteroatom, or an    optionally substituted group selected from the group consisting of    acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and    heterocyclic;-   each occurrence of T is independently a covalent bond or a bivalent,    straight or branched, saturated or unsaturated, optionally    substituted C₁₋₃₀ hydrocarbon chain wherein one or more methylene    units of T are optionally and independently replaced by —O—, —S—,    —N(R)—, —C(O)—, —C(O)O—, —OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—,    —S(O)₂—, —N(R)SO₂—, —SO₂N(R)—, a heterocyclic group, an aryl group,    or a heteroaryl group;-   each occurrence of R is independently hydrogen, a suitable    protecting group, or an acyl moiety, arylalkyl moiety, aliphatic    moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety;-   —B is -T-L^(B)-X;-   each occurrence of X is independently a ligand;-   each occurrence of L^(B) is independently a covalent bond or a group    derived from the covalent conjugation of a T with an X; and,-   wherein n is 1, 2, or 3, with the proviso that the insulin is    conjugated to at least one linker in which one of the ligands is    Fucose.

In particular aspects, the aforementioned conjugate may be characterizedas having a ratio of EC₅₀ or IP as determined by a functional insulinreceptor phosphorylation assay verses the IC₅₀ or IP as determined by acompetition binding assay at the macrophage mannose receptor is about0.5:1 to about 1:100; about 1:1 to about 1:50; about 1:1 to about 1:20;or about 1:1 to about 1:10.

In particular embodiments, the insulin or insulin analog conjugate mayhave the general formula (II):

wherein:

-   each occurrence of

represents a potential repeat within a branch of the conjugate;

-   each occurrence of    is independently a covalent bond, a carbon atom, a heteroatom, or an    optionally substituted group selected from the group consisting of    acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and    heterocyclic;-   each occurrence of T is independently a covalent bond or a bivalent,    straight or branched, saturated or unsaturated, optionally    substituted C₁₋₃₀ hydrocarbon chain wherein one or more methylene    units of T are optionally and independently replaced by —O—, —S—,    —N(R)—, —C(O)—, —C(O)O—, —OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—,    —S(O)₂—, —N(R)SO₂—, —SO₂N(R)—, a heterocyclic group, an aryl group,    or a heteroaryl group;-   each occurrence of R is independently hydrogen, a suitable    protecting group, or an acyl moiety, arylalkyl moiety, aliphatic    moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety;-   —B₁ is -T-L^(B1)-Fucose-   wherein L^(B1) is a covalent bond or a group derived from the    covalent conjugation of a T with an X;-   —B₂ is -T-L^(B2)-X-   wherein X is a ligand comprising a saccharide, which may be fucose,    mannose, or glucose; and L^(B2) is a covalent bond or a group    derived from the covalent conjugation of a T with an X; and,-   wherein n is 1, 2, or 3.

In particular aspects, the aforementioned conjugate may be characterizedas having a ratio of EC₅₀ or IP as determined by a functional insulinreceptor phosphorylation assay verses the IC₅₀ or IP as determined by acompetition binding assay at the macrophage mannose receptor is about0.5:1 to about 1:100; about 1:1 to about 1:50; about 1:1 to about 1:20;or about 1:1 to about 1:10.

Description of Exemplary Groups

(node)

In particular embodiments, each occurrence of

is independently an optionally substituted group selected from the groupconsisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, andheterocyclic. In some embodiments, each occurrence of

is the same. In some embodiments, the central

is different from all other occurrences of

. In particular embodiments, all occurrences of

are the same except for the central

.

In some embodiments,

is an optionally substituted aryl or heteroaryl group. In someembodiments,

is a 2-, 3, 4, 6, or 8-membered aryl or heteroaryl group. In someembodiments,

is a 5- or 6-membered heterocyclic group. In particular embodiments,

is a heteroatom selected from N, O, or S. In some embodiments,

is nitrogen atom. In some embodiments,

is an oxygen atom. In some embodiments,

is sulfur atom. In some embodiments,

is a carbon atom. In some embodiments,

is the structure

T (Spacer)

In particular embodiments, each occurrence of T is independently abivalent, straight or branched, saturated or unsaturated, optionallysubstituted C₁₋₂₀ hydrocarbon chain wherein one or more methylene unitsof T are optionally and independently replaced by —O—, —S—, —N(R)—,—C(O)—, —C(O)O—, —OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)₂—,—N(R)SO₂—, —SO₂N(R)—, a heterocyclic group, an aryl group, or aheteroaryl group. In particular embodiments, one, two, three, four, orfive methylene units of T are optionally and independently replaced. Inparticular embodiments, T is constructed from a C₁₋₁₀, C₁₋₈, C₁₋₆, C₁₋₄,C₂₋₁₂, C₄₋₁₂, C₆₋₁₂, C₈₋₁₂, or C₁₀₋₁₂ hydrocarbon chain wherein one ormore methylene units of T are optionally and independently replaced by—O—, —S—, —N(R)—, —C(O)—, —C(O)O—, —OC(O)—, —N(R)C(O)—, —C(O)N(R)—,—S(O)—, —S(O)₂—, —N(R)SO₂—, —SO₂N(R)—, a heterocyclic group, an arylgroup, or a heteroaryl group. In some embodiments, one or more methyleneunits of T is replaced by a heterocyclic group. In some embodiments, oneor more methylene units of T is replaced by a triazole moiety. Inparticular embodiments, one or more methylene units of T is replaced by—C(O)—. In particular embodiments, one or more methylene units of T isreplaced by —C(O)N(R)—. In particular embodiments, one or more methyleneunits of T is replaced by —O—.

In particular embodiments, T may be structure

In particular embodiments, the present disclosure provides insulin orinsulin analog conjugates comprising 1, 2, or 3 bi-dentate linkers, eachindependently selected from the group consisting of

wherein each X is independently a ligand comprising a saccharide withthe proviso that at least one bi-dentate linker conjugated to theinsulin or insulin analog comprises a fucose on at least one arm of thebi-dentate linker. In particular embodiments, each X may independentlybe

wherein the wavy line indicates the bond is linked to an atom comprisingthe bi-dentate linker. EG is ethylglucose, EM is ethylmannose, EF isethylfucose, ETM is ethyltrimannose, EBM is ethyldimannose, EGA isethylgluccosamine, EDG is ethyldeoxyglucose, EDF is ethyldeoxyfucose,and EDM is ethyldeoxymannose.

One of ordinary skill will appreciate that a variety of conjugationchemistries may be used to covalently conjugate an X with a T and/or a Wwith a T (generally “components”). Such techniques are widely known inthe art, and exemplary techniques are discussed below. Components can bedirectly bonded (i.e., with no intervening chemical groups) orindirectly bonded through a spacer (e.g., a coupling agent or covalentchain that provides some physical separation between the conjugatedelement and the remainder of the linker). It is to be understood thatcomponents may be covalently bound to a linker through any number ofchemical bonds, including but not limited to amide, amine, ester, ether,thioether, isourea, imine, etc. bonds.

In various embodiments, components may be covalently bound to a linkerusing “click chemistry” reactions as is known in the art. These include,for example, cycloaddition reactions, nucleophilic ring-openingreactions, and additions to carbon-carbon multiple bonds (e.g., see Kolband Sharpless, Drug Discovery Today 8:1128-1137, 2003 and referencescited therein as well as Dondoni, Chem. Asian J. 2:700-708, 2007 andreferences cited therein). As discussed above, in various embodiments,the components may be bound to a linker via natural or chemically addedpendant groups. In general, it will be appreciated that the first andsecond members of a pair of reactive groups (e.g., a carboxyl group andan amine group which react to produce an amide bond) can be present oneither one of the component and linker (i.e., the relative location ofthe two members is irrelevant as long as they react to produce aconjugate). Exemplary linkages are discussed in more detail below.

Particular components may naturally possess more than one of the samechemically reactive moiety. In some examples, it is possible to choosethe chemical reaction type and conditions to selectively react thecomponent at only one of those sites. For example, in the case whereinsulin is conjugated through reactive amines, in particularembodiments, the N-terminal α-Phe-B1 may be more desirable as a site ofattachment over the N-terminal α-Gly-A1 and ε-Lys-B29 to preserveinsulin bioactivity (e.g., see Mei et al., Pharm. Res. 16: 1680-1686,1999 and references cited therein as well as Tsai et al., J. Pharm. Sci.86: 1264-1268, 1997). In an exemplary reaction between insulin withhexadecenal (an aldehyde-terminated molecule), researchers found thatmixing the two components overnight in a 1.5M pH 6.8 sodium salicylateaqueous solution containing 54% isopropanol at a ratio of 1:6(insulin:aldehyde mol/mol) in the presence of sodium cyanoborohydrideresulted in over 80% conversion to the single-substituted Phe-B1secondary amine-conjugated product (Mei et al., Pharm. Res.16:1680-1686, 1999). Their studies showed that the choice of solvent,pH, and insulin:aldehyde ratio all affected the selectivity and yield ofthe reaction. In most cases, however, achieving selectivity throughchoice of chemical reaction conditions is difficult. Therefore, inparticular embodiments it may be advantageous to selectively protect thecomponent (e.g., insulin) at all sites other than the one desired forreaction followed by a deprotection step after the material has beenreacted and purified. For example, there are numerous examples ofselective protection of insulin amine groups available in the literatureincluding those that may be deprotected under acidic (BOC), slightlyacidic (citraconic anhydride), and basic (MSC) conditions (e.g., seeTsai et al., J. Pharm. Sci. 86: 1264-1268, 1997; Dixon et al., Biochem.J. 109: 312-314, 1968; and Schuettler et al., D. Brandenburg HoppeSeyler's Z. Physiol. Chem. 360: 1721, 1979). In one example, the Gly-A1and Lys-B29 amines may be selectively protected with tert-butoxycarbonyl(BOC) groups which are then removed after conjugation by incubation forone hour at 4 C in a 90% trifluoroacetic acid (TFA)/10% anisolesolution. In one embodiment, a dry powder of insulin is dissolved inanhydrous DMSO followed by an excess of triethylamine To this solution,approximately two equivalents of di-tert-butyl dicarbonate solution inTHF are added slowly and the solution allowed to mix for 30 to 60minutes. After reaction, the crude solution is poured in an excess ofacetone followed by dropwise addition of dilute HCl to precipitate thereacted insulin. The precipitated material is centrifuged, washed withacetone and dried completely under vacuum.

The desired di-BOC protected product may be separated from unreactedinsulin, undesired di-BOC isomers, and mono-BOC and tri-BOC byproductsusing preparative reverse phase HPLC or ion exchange chromatography(e.g., see Tsai et al., J. Pharm. Sci. 86: 1264-1268, 1997). In the caseof reverse phase HPLC, a solution of the crude product in 70% water/30%acetonitrile containing 0.1% TFA is loaded onto a C8 column and elutedwith an increasing acetonitrile gradient. The desired di-BOC peak iscollected, the acetonitrile removed and lyophilized to obtain theproduct.

In particular embodiments, the insulin oligosaccharide conjugates maycomprise an insulin or insulin analog and at least one bi-dentate linkerhaving a first arm and second arm, wherein the first arm is linked to afirst ligand that includes a first saccharide and the second arm islinked to a second ligand that includes a second saccharide and whereinfor at least one bi-dentate linker the first saccharide is fucose, andwherein the at least one bi-dentate linker is conjugated to the alphaamino group of the N-terminal amino acid of the A-chain or B-chain ofthe insulin or insulin analog or to the epsilon amino group of a lysineresidue of the A-chain or the B-chain of the insulin or insulin analog.In particular embodiments the conjugate may include at least two linkerswherein at least one linker is a bidentate linker comprising a fucose.In particular embodiments the conjugate may include at least threelinkers wherein at least one linker is a bidentate linker comprising afucose.

In particular embodiments, the at least one bi-dentate linker may haveformula A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U,V, W, X, Y, Z, AA, AB, AC, AD, AE, AF, AG, AH, AI, AJ, or AK as shownsupra wherein X is a saccharide; with the proviso that for at least onebi-dentate linker the X on at least one arm of the at least onebi-dentate linker is fucose. In particular embodiments, X has theformula EG, EM, EBM, EGA, EF, EFβ, EBM, ETM, EDG, EDF, or EDM as shownsupra.

In particular embodiments, the insulin analog may comprise an A chainsequence comprising a sequence of GIVEQCCX₁SICSLYQLENYCX₂ (SEQ ID NO:8); and a B chain sequence comprising a sequence of X₃LCGX₄X₅LVEALYLVCGERGFF (SEQ ID NO: 9) or X₈VNQX₃LCGX₄X₅LVEALYLVCGERGFFYTX₆X₇ (SEQ ID NO:10) wherein

X₁ is selected from the group consisting of threonine and histidine;

X₂ is asparagine or glycine;

X₃ is selected from the group consisting of histidine and threonine;

X₄ is selected from the group consisting of alanine, glycine and serine;

X₅ is selected from the group consisting of histidine, aspartic acid,glutamic acid, homocysteic acid and cysteic acid;

X₆ is aspartate-lysine dipeptide, a lysine-proline dipeptide, or aproline-lysine dipeptide;

X₇ is threonine, alanine, or a threonine-arginine-arginine tripeptide;and

X₈ is selected from the group consisting of phenylalanine anddesamino-phenylalanine.

In particular embodiments, the A-chain may have the amino acid sequenceset forth in SEQ ID NO:1 or SEQ ID NO:6 and the B-chain may have theamino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,or SEQ ID NO:5. In particular embodiments, the insulin analog is a desB30 insulin analog, a des B29-B30 insulin analog, a des B28-B30 insulinanalog, a des B27-B30 insulin analog or a des B26-B30 insulin analog.

In particular embodiments, the insulin oligosaccharide conjugate of thepresent invention may have the formula

Wherein R₁, R₂, and R₃, are each independently either H (hydrogen) or alinear linker having a ligand thereon that includes a saccharide or abi-dentate linker having a first arm and second arm, wherein the firstarm is linked to a first ligand that includes a first saccharide and thesecond arm is linked to a second ligand that includes a secondsaccharide with the proviso that at least one of R₁, R₂, or R₃ is abi-dentate linker wherein the first saccharide is fucose. In aparticular embodiment, R₁, R₂, or R₃ are each independently either H(hydrogen) or a bi-dentate linker having formula A, B, C, D, E, F, G, H,I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y, Z, AA, AB, AC, AD,AE, AF, AG, AH, AI, AJ, or AK as shown supra wherein X is a saccharide;with the proviso that at least one of R₁, R₂, or R₃ is a bi-dentatelinker and the X on at least one arm of at least one bi-dentate linkeris fucose. In particular embodiments, X has the formula EG, EM, EBM,EGA, EF, EFβ, EBM, ETM, EDG, EDF, or EDM as shown supra.

Exemplary human insulin oligosaccharide conjugates (IOCs) of the presentinvention include the IOCs having the following structures.

Sustained Release Formulations

In particular embodiments it may be advantageous to administer aninsulin conjugate in a sustained fashion (i.e., in a form that exhibitsan absorption profile that is more sustained than soluble recombinanthuman insulin). This will provide a sustained level of conjugate thatcan respond to fluctuations in glucose on a timescale that it moreclosely related to the typical glucose fluctuation timescale (i.e.,hours rather than minutes). In particular embodiments, the sustainedrelease formulation may exhibit a zero-order release of the conjugatewhen administered to a mammal under non-hyperglycemic conditions (i.e.,fasted conditions).

It will be appreciated that any formulation that provides a sustainedabsorption profile may be used. In particular embodiments this may beachieved by combining the conjugate with other ingredients that slow itsrelease properties into systemic circulation. For example, PZI(protamine zinc insulin) formulations may be used for this purpose. Thepresent disclosure encompasses amorphous and crystalline forms of thesePZI formulations.

Thus, in particular embodiments, a formulation of the present disclosureincludes from about 0.05 to about 10 mg protamine/mg conjugate. Forexample, from about 0.2 to about 10 mg protamine/mg conjugate, e.g.,about 1 to about 5 mg protamine/mg conjugate.

In particular embodiments, a formulation of the present disclosureincludes from about 0.006 to about 0.5 mg zinc/mg conjugate. Forexample, from about 0.05 to about 0.5 mg zinc/mg conjugate, e.g., about0.1 to about 0.25 mg zinc/mg conjugate.

In particular embodiments, a formulation of the present disclosureincludes protamine and zinc in a ratio (w/w) in the range of about 100:1to about 5:1, for example, from about 50:1 to about 5:1, e.g., about40:1 to about 10:1. In particular embodiments, a PZI formulation of thepresent disclosure includes protamine and zinc in a ratio (w/w) in therange of about 20:1 to about 5:1, for example, about 20:1 to about 10:1,about 20:1 to about 15:1, about 15:1 to about 5:1, about 10:1 to about5:1, about 10:1 to about 15:1.

One or more of the following components may be included in the PZIformulation: an antimicrobial preservative, an isotonic agent, and/or anunconjugated insulin molecule.

In particular embodiments a formulation of the present disclosureincludes an antimicrobial preservative (e.g., m-cresol, phenol,methylparaben, or propylparaben). In particular embodiments theantimicrobial preservative is m-cresol. For example, in particularembodiments, a formulation may include from about 0.1 to about 1.0% v/vm-cresol. For example, from about 0.1 to about 0.5% v/v m-cresol, e.g.,about 0.15 to about 0.35% v/v m-cresol.

In particular embodiments a formulation of the present disclosureincludes a polyol as isotonic agent (e.g., mannitol, propylene glycol orglycerol). In particular embodiments the isotonic agent is glycerol. Inparticular embodiments, the isotonic agent is a salt, e.g., NaCl. Forexample, a formulation may comprise from about 0.05 to about 0.5 M NaCl,e.g., from about 0.05 to about 0.25 M NaCl or from about 0.1 to about0.2 M NaCl.

In particular embodiments a formulation of the present disclosureincludes an amount of unconjugated insulin molecule. In particularembodiments, a formulation includes a molar ratio of conjugated insulinmolecule to unconjugated insulin molecule in the range of about 100:1 to1:1, e.g., about 50:1 to 2:1 or about 25:1 to 2:1.

The present disclosure also encompasses the use of standard sustained(also called extended) release formulations that are well known in theart of small molecule formulation (e.g., see Remington's PharmaceuticalSciences, 19^(th) ed., Mack Publishing Co., Easton, Pa., 1995). Thepresent disclosure also encompasses the use of devices that rely onpumps or hindered diffusion to deliver a conjugate on a gradual basis.In particular embodiments, a long acting formulation may (additionallyor alternatively) be provided by using a modified insulin molecule. Forexample, one could use insulin glargine (LANTUS®) or insulin detemir(LEVEMIR®) instead of wild-type human insulin in preparing theconjugate. Insulin glargine is an exemplary long acting insulin analogin which Asn at position A21 of the A-chain has been replaced by glycineand two arginine residues are at the C-terminus of the B-chain. Theeffect of these changes is to shift the isoelectric point, producing aninsulin that is insoluble at physiological pH but is soluble at pH 4.Insulin detemir is another long acting insulin analog in which Thr atposition B30 of the B-chain has been deleted and a C14 fatty acid chainhas been attached to the Lys at position B29.

Uses of Conjugates

In another aspect, the present disclosure provides methods of using theinsulin conjugates. In general, the insulin conjugates can be used tocontrollably provide insulin to an individual in need in response to asaccharide (e.g., glucose or an exogenous saccharide such as mannose,alpha-methyl mannose, L-fucose, etc.). The disclosure encompassestreating diabetes by administering an insulin conjugate of the presentdisclosure. Although the insulin conjugates can be used to treat anypatient (e.g., dogs, cats, cows, horses, sheep, pigs, mice, etc.), theyare most preferably used in the treatment of humans. An insulinconjugate may be administered to a patient by any route. In general, thepresent disclosure encompasses administration by oral, intravenous,intramuscular, intra-arterial, subcutaneous, intraventricular,transdermal, rectal, intravaginal, intraperitoneal, topical (as bypowders, ointments, or drops), buccal, or as an oral or nasal spray oraerosol. General considerations in the formulation and manufacture ofpharmaceutical compositions for these different routes may be found, forexample, in Remington's Pharmaceutical Sciences, 19^(th) ed., MackPublishing Co., Easton, Pa., 1995. In various embodiments, the conjugatemay be administered subcutaneously, e.g., by injection. The insulinconjugate may be dissolved in a carrier for ease of delivery. Forexample, the carrier can be an aqueous solution including, but notlimited to, sterile water, saline or buffered saline.

In general, a therapeutically effective amount of the insulin conjugatewill be administered. The term “therapeutically effective amount” meansa sufficient amount of the insulin conjugate to treat diabetes at areasonable benefit/risk ratio, which involves a balancing of theefficacy and toxicity of the insulin conjugate. In various embodiments,the average daily dose of insulin is in the range of 10 to 200 U, e.g.,25 to 100 U (where 1 Unit of insulin is ˜0.04 mg). In particularembodiments, an amount of conjugate with these insulin doses isadministered on a daily basis. In particular embodiments, an amount ofconjugate with 5 to 10 times these insulin doses is administered on aweekly basis. In particular embodiments, an amount of conjugate with 10to 20 times these insulin doses is administered on a bi-weekly basis. Inparticular embodiments, an amount of conjugate with 20 to 40 times theseinsulin doses is administered on a monthly basis.

In particular embodiments, a conjugate of the present disclosure may beused to treat hyperglycemia in a patient (e.g., a mammalian or humanpatient). In particular embodiments, the patient is diabetic. However,the present methods are not limited to treating diabetic patients. Forexample, in particular embodiments, a conjugate may be used to treathyperglycemia in a patient with an infection associated with impairedglycemic control. In particular embodiments, a conjugate may be used totreat diabetes.

In particular embodiments, when an insulin conjugate or formulation ofthe present disclosure is administered to a patient (e.g., a mammalianpatient) it induces less hypoglycemia than an unconjugated version ofthe insulin molecule. In particular embodiments, a formulation of thepresent disclosure induces a lower HbA1c value in a patient (e.g., amammalian or human patient) than a formulation comprising anunconjugated version of the insulin molecule. In particular embodiments,the formulation leads to an HbA1c value that is at least 10% lower(e.g., at least 20% lower, at least 30% lower, at least 40% lower, atleast 50% lower) than a formulation comprising an unconjugated versionof the insulin molecule. In particular embodiments, the formulationleads to an HbA1c value of less than 7%, e.g., in the range of about 4to about 6%. In particular embodiments, a formulation comprising anunconjugated version of the insulin molecule leads to an HbA1c value inexcess of 7%, e.g., about 8 to about 12%.

Exogenous Trigger

As mentioned previously, the methods, conjugates and compositions thatare described herein are not limited to glucose responsive-conjugates.As demonstrated in the Examples, several exemplary insulin conjugateswere also responsive to exogenous saccharides such as alpha-methylmannose. It will therefore be appreciated that in particular embodimentsan insulin conjugate may be triggered by exogenous administration of asaccharide other than glucose such as alpha-methyl mannose or any othersaccharide that can alter the PK or PD properties of the conjugate.

Once a conjugate has been administered as described above (e.g., as asustained release formulation) it can be triggered by administration ofa suitable exogenous saccharide. In a particular embodiment, atriggering amount of the exogenous saccharide is administered. As usedherein, a “triggering amount” of exogenous saccharide is an amountsufficient to cause a change in at least one PK and/or PD property ofthe conjugate (e.g., C_(max), AUC, half-life, etc. as discussedpreviously). It is to be understood that any of the aforementionedmethods of administration for the conjugate apply equally to theexogenous saccharide. It is also be to be understood that the methods ofadministration for the conjugate and exogenous saccharide may be thesame or different. In various embodiments, the methods of administrationare different (e.g., for purposes of illustration the conjugate may beadministered by subcutaneous injection on a weekly basis while theexogenous saccharide is administered orally on a daily basis). The oraladministration of an exogenous saccharide is of particular value sinceit facilitates patient compliance. In general, it will be appreciatedthat the PK and PD properties of the conjugate will be related to the PKprofile of the exogenous saccharide. Thus, the conjugate PK and PDproperties can be tailored by controlling the PK profile of theexogenous saccharide. As is well known in the art, the PK profile of theexogenous saccharide can be tailored based on the dose, route, frequencyand formulation used. For example, if a short and intense activation ofthe conjugate is desired then an oral immediate release formulationmight be used. In contrast, if a longer less intense activation ofconjugate is desired then an oral extended release formulation might beused instead. General considerations in the formulation and manufactureof immediate and extended release formulation may be found, for example,in Remington's Pharmaceutical Sciences, 19^(th) ed., Mack PublishingCo., Easton, Pa., 1995.

It will also be appreciated that the relative frequency ofadministration of a conjugate of the present disclosure and an exogenoussaccharide may be the same or different. In particular embodiments, theexogenous saccharide is administered more frequently than the conjugate.For example, in particular embodiment, the conjugate may be administereddaily while the exogenous saccharide is administered more than once aday. In particular embodiment, the conjugate may be administered twiceweekly, weekly, biweekly or monthly while the exogenous saccharide isadministered daily. In particular embodiments, the conjugate isadministered monthly and the exogenous saccharide is administered twiceweekly, weekly, or biweekly. Other variations on these schemes will berecognized by those skilled in the art and will vary depending on thenature of the conjugate and formulation used.

The following examples are intended to promote a further understandingof the present invention.

EXAMPLES

General Procedures

All chemicals were purchased from commercial sources, unless otherwisenoted. Reactions sensitive to moisture or air were performed undernitrogen or argon using anhydrous solvents and reagents. The progress ofreactions was monitored by analytical thin layer chromatography (TLC),high performance liquid chromatography-mass spectrometry (HPLC-MS), orultra performance liquid chromatography-mass spectrometry (UPLC-MS). TLCwas performed on E. Merck TLC plates precoated with silica gel 60F-254,layer thickness 0.25 mm. The plates were visualized using 254 nm UVand/or by exposure to cerium ammonium molybdate (CAM) or p-anisaldehydestaining solutions followed by charring. High performance liquidchromatography (HPLC) was conducted on an Agilent 1100 series HPLC usingSupelco Ascentis Express C18 2.7 μm 3.0×100 mm column with gradient10:90-99:1 v/v CH₃CN/H₂O+v 0.05% TFA over 4.0 min then hold at 98:2 v/vCH₃CN/H₂O+v 0.05% TFA for 0.75 min; flow rate 1.0 mL/min, UV range200-400 nm (LC-MS Method A). Mass analysis was performed on a WatersMicromass® ZQ™ with electrospray ionization in positive ion detectionmode and the scan range of the mass-to-charge ratio was either 170-900or 500-1500. Ultra performance liquid chromatography (UPLC) wasperformed on a Waters Acquity™ UPLC® system using Waters Acquity™ UPLC®BEH300 C4 1.7 μm 2.1×100 mm column with gradient 10:90-90:10 v/vCH₃CN/H₂O+v 0.1% TFA over 4.0 min and 90:10-95:5 v/v CH₃CN/H₂O+v 0.1%TFA over 0.5 min; flow rate 0.3 mL/min, UV wavelength 200-300 nm (UPLCMethod A). Alternative UPLC conditions were noted as UPLC Method B(Waters Acquity™ UPLC® BEH C18 1.7 μm 2.1×100 mm column with gradient10:90-70:30 v/v CH₃CN/H₂O+v 0.1% TFA over 4.0 min and 70:30-95:5 v/vCH₃CN/H₂O+v 0.1% TFA over 40 sec; flow rate 0.3 mL/min, UV wavelength200-300 nm), UPLC Method C (Waters Acquity™ UPLC® BEH C18 1.7 μm 2.1×100mm column with gradient 60:40-100:0 v/v CH₃CN/H₂O+v 0.1% TFA over 4.0min and 100:0-95:5 v/v CH₃CN/H₂O+v 0.1% TFA over 40 sec; flow rate 0.3mL/min, UV wavelength 200-300 nm), UPLC Method D (Waters Acquity™ UPLC®BEH300 C4 1.7 μm 2.1×100 mm column with gradient 10:90-50:50 v/vCH₃CN/H₂O+v 0.1% TFA over 4.3 min and 50:50-70:30 v/v CH₃CN/H₂O+v 0.1%TFA over 0.5 min; flow rate 0.3 mL/min, UV wavelength 200-300 nm), UPLCMethod E (Waters Acquity™ UPLC® BEH C18 1.7 μm 2.1×100 mm column withgradient 10:90-60:40 v/v CH₃CN/H₂O+v 0.1% TFA over 4.3 min and60:40-90:10 v/v CH₃CN/H₂O+v 0.1% TFA over 0.5 min; flow rate 0.3 mL/min,UV wavelength 200-300 nm), UPLC Method F (Waters Acquity™ UPLC® BEH C181.7 μm 2.1×100 mm column with gradient 60:40-100:0 v/v CH₃CN/H₂O+v 0.1%TFA over 4.0 min and 100: 0-95:5 v/v CH₃CN/H₂O+v 0.1% TFA over 0.4 min;flow rate 0.3 mL/min, UV wavelength 200-300 nm), and UPLC Method G(Waters Acquity™ UPLC® BEH C8 1.7 μm 2.1×100 mm column with gradient10:90-55:45 v/v CH₃CN/H₂O+v 0.1% TFA over 4.2 min and 100: 0-95:5 v/vCH₃CN/H₂O+v 0.1% TFA over 0.4 min; flow rate 0.3 mL/min, UV wavelength200-300 nm. Mass analysis was performed on a Waters Micromass® LCTPremier™ XE with electrospray ionization in positive ion detection modeand the scan range of the mass-to-charge ratio was 300-2000. Theidentification of the produced insulin conjugates was confirmed bycomparing the theoretical molecular weight to the experimental valuethat was measured using UPLC-MS. For the determination of the positionof sugar modification(s), specifically, insulin conjugates weresubjected to DTT treatment (for a/b chain) or Glu-C digestion (withreduction and alkylation), and then the resulting peptides were analyzedby LC-MS. Based on the measured masses, the sugar positions werededuced.

Flash chromatography was performed using either a Biotage FlashChromatography apparatus (Dyax Corp.) or a CombiFlash® Rf instrument(Teledyne Isco). Normal-phase chromatography was carried out on silicagel (20-70 μm, 60 Å pore size) in pre-packed cartridges of the sizenoted. Reverse-phase chromatography was carried out on C18-bonded silicagel (20-60 μm, 60-100 Å pore size) in pre-packed cartridges of the sizenoted. Preparative scale HPLC was performed on Gilson 333-334 binarysystem using Waters Delta Pak C4 15 μm, 300 Å, 50×250 mm column orKromasil® C8 10 μm, 100 Å, 50×250 mm column, flow rate 85 mL/min, withgradient noted. Concentration of solutions was carried out on a rotaryevaporator under reduced pressure or freeze-dried on a VirTisFreezemobile Freeze Dryer (SP Scientific).

¹H NMR spectra were acquired at 500 MHz (or otherwise specified)spectrometers in deuterated solvents noted. Chemical shifts werereported in parts per million (ppm). Tetramethylsilane (TMS) or residualproton peak of deutrated solvents was used as an internal reference.Coupling constant (J) were reported in hertz (Hz).

Abbreviations: acetic acid (AcOH), acetonitrile (AcCN), aqueous (aq),O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate) (HATU), ethyl acetate (EtOAc), diethyl ether (etheror Et₂O), N,N-diisopropylethylamine or Hünig's base (DIPEA),(4-dimethylamino)pyridine (DMAP), N,N-dimethylformamide (DMF), ethylacetate (EtOAc), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC), gram(s) (g), 1-hydroxybenzotriazole hydrate (HOBt),hour(s) (h or hr), mass spectrum (ms or MS), microliter(s) (μL),milligram(s) (mg), milliliter(s) (mL), millimole (mmol), minute(s)(min), pentafluorphenol-tetramethyluronium hexafluorophosphate (PFTU),petroleum ether (PE), retention time (R_(t)), room temperature (rt),saturated (sat. or sat'd), saturated aq sodium chloride solution(brine), triethylamine (TEA), trifluoroacetic acid (TFA),tetrahydrofuran (THF), andN,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate(TSTU).

Example 1

The synthesis of oligosaccharide linker6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)-6-oxohexanamide(ML-1) having the following structure is described.

Step A: benzyl 6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexanoate

To a solution of 6-(benzyloxy)-6-oxohexanoic acid (3.3 g, 13.97 mmol) inDMF (50 mL) at 0° C. was added TSTU (4.3 g, 14.28 mmol) and DIPEA (2.5mL, 14.31 mmol). After stirring at 0° C. for 1 hour, the reactionmixture was partitioned between Et₂O and water. The organic layer wasseparated and the aqueous layer was further extracted with ether (2×150mL). The combined organic phase was washed with brine, dried overNa₂SO₄, filtered and concentrated to afford the title compound. UPLCMethod B: calculated for C₁₇H₁₉NO₆ 333.12, observed m/e: 334.10 [M+1];Rt=3.75 min. ¹H NMR (CDCl₃) δ 7.40-7.30 (5H, m), 5.10 (2H, s), 2.80 (4H,s), 2.62-2.58 (2H, m), 2.41-2.37 (2H, m), 1.80-1.72 (4H, m).

Step B: benzyl6-({2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}amino)-6-oxohexanoate

To a solution of 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranoside(1.23 g, 2.247 mmol, WO 2010/088294 A1) in DMF (20 mL) at 0° C. wasadded benzyl 6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexanoate (1.02 g,3.06 mmol) and TEA (0.5 mL, 3.59 mmol). After stirring at 0° C. for 1hour, the reaction mixture was concentrated and the residue was purifiedby flash chromatography on C18 reverse silica gel column (275 g),eluting with 0-40% AcCN in H₂O to give the title compound. UPLC MethodB: calculated for C₃₃H₅₁NO₁₉ 765.31, observed m/e=766.26 [M+1]; Rt=4.04min. ¹H NMR (D₂O) δ 7.43-7.37 (5H, m), 5.14 (2H, s), 5.07-5.06 (1H, m),4.82-4.81 (1H, m), 4.77-4.76 (1H, m), 4.06-4.01 (2H, m), 3.96-3.92 (2H,m), 3.87-3.81 (5H, m), 3.79-3.77 (1H, m), 3.74-3.67 (5H, m), 3.65-3.60(4H, m), 3.53-3.49 (1H, m), 3.37-3.35 (2H, m), 2.43-2.40 (2H, m),2.22-2.19 (2H, m), 1.62-1.52 (4H, m).

Step C:6-({2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}amino)-6-oxohexanoicacid

A mixture of benzyl6-({2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}amino)-6-oxohexanoate(1.15 g, 1.502 mmol) and Pd/C (80 mg, 0.075 mmol) in water (10 mL) wasallowed to stir under a balloon of H₂ at room temperature for 16 hr. Thecatalyst was filtered off and washed with H₂O (3×10 mL). The filtratewas concentrated to give the title compound. UPLC Method B: calculatedfor C₂₆H₄₅NO₁₉ 675.26, observed m/e: 676.21 [M+1]; Rt=3.50 min.

Step D:6-[(2,5-dioxopyrrolidin-1-yl)oxy]-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)-6-oxohexanamide

To a solution of6-({2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}amino)-6-oxohexanoicacid (1.55 g, 2.294 mmol) in DMF (22 mL) at 0° C. was added TSTU (760mg, 2.52 mmol) and DIPEA (0.52 mL, 2.98 mmol). After stirring at 0° C.for 1 hr, the reaction was quenched by the addition of TFA (371 μL, 4.82mmol) and the resulting mixture was concentrated down to about 3 mL. Theresidue was transferred dropwise, via autopipette, to a tube containinganhydrous acetonitrile (45 mL). The white precipitate was collectedthrough centrifugation (3000 rpm, 15 min, at 4° C.), washed withanhydrous AcCN (1 mL) and dried to yield the title compound. UPLC MethodB: calculated for C₃₀H₄₈N₂O₂₁ 772.27, observed m/e: 773.23 [M+1];Rt=3.65 min. ¹H NMR (D₂O) δ 5.07-5.06 (1H, m), 4.84-4.83 (1H, m),4.79-4.78 (1H, m), 4.06-4.01 (2H, m), 3.96-3.93 (2H, m), 3.87-3.83 (5H,m), 3.80-3.78 (1H, m), 3.75-3.69 (5H, m), 3.67-3.61 (4H, m), 3.57-3.52(1H, m), 3.41-3.38 (2H, m), 2.91 (4H, s), 2.75-2.71 (2H, m), 2.29-2.25(2H, m), 1.75-1.58 (4H, m).

Example 2

The synthesis of oligosaccharide linker6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-N-(2-{[3-O-(α-D-mannopyranosyl)-α-D-mannopyranosyl]oxy}ethyl)-6-oxohexanamide(ML-2) having the following structure is described.

Step A: 2-azidoethyl 2,4-di-O-benzoyl-6-O-trityl-β-D-mannopyranoside

In a 250 ml round bottom flask, 2-azidoethyl2,4-di-O-benzoyl-α-D-mannopyranoside (1.0 g, 2.186 mmol; See WO2010/088294 A1, incorporated herein by reference) was dissolved inpyridine (50 mL). To the above solution was added DMAP (13 mg, 0.109mmol) followed by trityl chloride (762 mg, 2.73 mmol). After stirring at80° C. for 18 hr, the reaction mixture was concentrated. The residue waspurified by flash chromatography on silica gel (40 g), eluting with0-50% EtOAc in hexanes to give the title compound. UPLC Method C:m/e=722.2955, [M+Na]; Rt=4.50. ¹H NMR (CDCl₃) δ 7.0-8.3 (m, 25H), 5.8(t, 1H), 5.5(m, 1H), 5.2 (s, 1H), 4.3 (m, 1H), 4.1 (m, 2H), 4.0 (m, 1H),3.5 (m, 1H), 3.4 (m, 2H), 3.2 (dd, 1H), 2.7 (d, 1H).

Step B: 2-azidoethyl2,4-di-O-benzoyl-3-O-(2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl)-6-O-trityl-α-D-mannopyranoside

In a 100 mL round bottom flask was added 2-azidoethyl2,4-bis-O-benzoyl-6-O-trityl-α-D-mannopyranoside (400 mg, 0.572 mmol),2,3,4,6-tetra-O-benzoyl-1-O-(2,2,2-trichloroethanimidoyl)-α-D-mannopyranose(508 mg, 0.686 mmol) and 4 Å molecular sieves (300 mg). To the abovemixture was added CH₂Cl₂ (5 mL). The reaction mixture was cooled to −78°C., to which was added TMSOTf (10.33 μL, 0.057 mmol). The mixture wasallowed to gradually warm to 0° C. and stirred for 30 min. The reactionwas then quenched with sat'd NaHCO₃, and filtered through a pad ofCelite. The filtrate was diluted with CH₂Cl₂ (20 mL), washed with brineand water. The organic phase was dried over MgSO₄, and concentrated. Theresidue was purified by flash chromatography on silica gel (80 g),eluting with 0-100% EtOAc in hexanes, to give the title product. LC-MSMethod A: m/e=1278.80 [M+1]; Rt=3.14 min. ¹H NMR (CDCl₃) δ 7.1-8.3 (m,30H), 6.0 (t, 1H), 5.8 (t, 1H), 5.7 (m, 2H), 5.4 (s, 1H), 5.38 (m, 1H),5.2 (s, 1H), 4.7 (dd, 1H), 4.6 (dd, 1H), 4.45 (m, 1H), 4.35 (dd, 1H),3.9-4.0 (m, 2H), 3.8 (m, 2H), 3.7 (m, 1H), 3.4 (m, 2H).

Step C: 2-azidoethyl2,4-di-O-benzoyl-3-O-(2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl)-α-D-mannopyranoside

In a 50 mL round bottom flask was added 2-azidoethyl2,4-di-O-benzoyl-3-O-(2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl)-6-O-trityl-α-D-mannopyranoside(450 mg, 0.352 mmol) and CH₂Cl₂ (3 mL). To the above solution was addedTFA (3 mL, 38.9 mmol). After stirring at 25° C. for 1 hr, the reactionmixture was diluted with CH₂Cl₂ (10 mL), washed with water (3×15 mL) andbrine (10 mL). The organic phase was dried over MgSO₄, filtered andconcentrated. The residue was purified by flash chromatography on silicagel (40 g), eluting with 0-100% EtOAc in hexanes, to give the titleproduct. LC-MS Method A: m/e=1053.57 [M+18]; Rt=2.73 min. ¹H NMR (CDCl₃)δ 7.0-8.5 (m, 45H), 6.1 (t, 1H), 6.0 (t, 1H), 5.7 (m, 2H), 5.4 (s, 1H),5.3 (s, 1H), 5.25 (s, 1H), 4.6 (m, 2H), 4.5 (m, 1H), 4.3 (m, 1H),4.0-4.2 (m, 3h), 3.8 (m, 1H), 3.3-3.5 (m, 3H).

Step D: 2-azidoethyl 3-O-α-D-mannopyranosyl-α-D-mannopyranoside

In a 50 mL round bottom flask was added 2-azidoethyl2,4-di-O-benzoyl-3-O-(2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl)-α-D-mannopyranoside(350 mg, 0.338 mmol) and CH₃OH (5 mL). To the above solution was addedNaOCH₃ (conc) dropwise till pH >10. The reaction mixture was allowed tostir at 25° C. for 6 hr. To the above solution was added DowexH+(50W×8-200) resin till pH ˜7. The solid resin was filtered off and thefiltrate was concentrated to give the title compound. LC-MS Method A:m/e=434.00 [M+1]; Rt=0.44 min.

Step E: 2-aminoethyl 3-O-α-D-mannopyranosyl-α-D-mannopyranoside

In a 50 mL round bottom flask, 2-azidoethyl3-O-α-D-mannopyranosyl-α-D-mannopyranoside (139 mg, 0.338 mmol) wasdissolved in water/CH₃OH (v/v 1:1, 5 mL). To the above solution wasadded Pd/C (10%, 36 mg, 0.034 mmol). The reaction mixture was stirred at25° C. under H₂ balloon for 18 hr. The mixture was filtered through apad of Celite, and the filtrate was concentrated to give the titlecompound. LC-MS Method A: m/e=386.08 [M+1]; Rt=0.24 min.

Step F:6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-N-(2-{[3-O-(α-D-mannopyranosyl)-α-D-mannopyranosyl]oxy}ethyl)-6-oxohexanamide

The title compound was prepared using procedures analogous to thosedescribed for ML-1 substituting 2-aminoethyl3-O-α-D-mannopyranosyl-α-D-mannopyranoside for 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosidein Step B. UPLC Method B: m/e=611.201 [M+1]: Rt=1.82 min.

Example 3

The synthesis of oligosaccharide linker6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-N-(2-{[6-O-(α-D-mannopyranosyl)-α-D-mannopyranosyl]oxy}ethyl)-6-oxohexanamide(ML-3) having the following structure is described.

Step A: 2-azidoethyl 2,3,4-tri-O-benzoyl-6-trityl-α-D-mannopyranoside

In a 250 mL round bottom flask, 2-azidoethyl2,4-di-O-benzoyl-α-D-mannopyranoside (1.0 g, 1.429 mmol) was dissolvedin pyridine (20 mL). To the above solution at 0° C. was added benzoylchloride (166 μL, 1.429 mmol). After stirring at rt for 18 hr, themixture was concentrated and the residue was dissolved in EtOAc (20 mL),washed with water (10 mL) and brine (10 mL). The organic phase was driedover MgSO₄, filtered and concentrated. The residue was purified by flashchromatography on silica gel (40 g), eluting with 0-50% EtOAc in hexane,to give the title compound. LC-MS Method A: m/e=804.44 [M+1]; Rt=2.88min. ¹H NMR (CDCl₃) δ 7.0-8.2 (m, 30H), 6.1 (t, 1H), 5.8 (dd, 1H), 5.2(d, 1H), 4.2-4.3 (m, 1H), 4.0-4.1 (m, 1H), 3.8 (m, 1H), 3.6 (m, 1H), 3.5(m, 1H), 3.4 (dd, H), 3.3 (dd, 1H).

Step B: 2-azidoethyl 2,3,4-tri-O-benzoyl-α-D-mannopyranoside

In a 100 mL round bottom flask, 2-azidoethyl2,3,4-tri-O-benzoyl-6-trityl-α-D-mannopyranoside (1.1 g, 1.368 mmol) wasdissolved in CH₂Cl₂ (10 mL). To the above solution was added TFA (10 mL,130 mmol). After stirring at 25° C. for 18 hr, the mixture was dilutedwith CH₂Cl₂ (20 mL), washed with brine (10 mL) and sat NaHCO₃ till pH˜7. The organic phase was dried over MgSO₄, filtered and concentrated.The residue was purified by flash chromatography on silica gel (40 g),eluting with 0-100% EtOAc in hexane, to to give the title compound.LC-MS Method A: m/e=579.12 [M+18] and 584.10 [M+Na]; Rt=2.22 min. ¹H NMR(CDCl₃) δ 7.2-8.2 (m, 15H), 6.0 (dd, 1H), 5.9 (t, 1H), 5.7 (m, 1H), 5.2(br-s, 1H), 4.1-4.2 (m, 2H), 3.85 (m, 1H), 3.7-3.8 (m, 2H), 3.6 (m, 1H,3.5 (m, 1H).

Step C: 2-azidoethyl2,3,4-tri-O-benzoyl-6-O-(2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl)-α-D-mannopyranoside

In a 100 mL round bottom flask was added 2-azidoethyl2,3,4-tri-O-benzoyl-α-D-mannopyranoside (720 mg, 1.282 mmol),2,3,4,6-tetra-O-benzoyl-1-O-(2,2,2-trichloroethanimidoyl)-α-D-mannopyranose(1.14 g, 1.539 mmol) and 4 Å molecular sieves (300 mg). To the abovemixture was added CH₂Cl₂ (10 mL). The reaction mixture was cooled to-78° C. To the above mixture was added TMSOTf (23.2 μL, 0.128 mmol). Themixture was allowed to gradually warm to 0° C. and stirred for 30 min.The reaction was then quenched with sat. NaHCO₃, and the mixture wasfiltered through a pad of Celite. The filtrate was diluted with CH₂Cl₂(20 mL), washed with brine and water. The organic phase was dried overMgSO₄, filtered and concentrated. The residue was purified by flashchromatography on silica gel (80 g), eluting with 0-100% EtOAc inhexanes, to give the title product. LC-MS Method A: m/e=1157.64 [M+18]and 1163.52 [M+Na]; Rt=3.01 min. ¹H NMR (CDCl₃) δ 7.2-8.3 (m, 25H), 6.0(t, 1H), 5.8 (t, 1H), 5.7 (m, 2H), 5.4 (s, 1H), 5.38 (m, 1H), 5.2 (s,1H), 4.7 (dd, 1H), 4.6 (dd, 1H), 4.45 (m, 1H), 4.35 (dd, 1H), 3.9-4.0(m, 2H), 3.8 (m, 2H), 3.7 (m, 1H), 3.4 (m, 2H).

Step D:6-[(2,5-dioxopyrrolidin-1-yl)oxy]-N-(2-{[6-O-(α-D-mannopyranosyl)-α-D-mannopyranosyl]oxy}ethyl)-6-oxohexanamide

The title compound was prepared using procedures analogous to thosedescribed for ML-2 substituting 2-azidoethyl2,3,4-tri-O-benzoyl-6-O-(2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl)-α-D-mannopyranosidefor 2-azidoethyl2,4-bis-O-benzoyl-3-O-(2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl)-α-D-mannopyranosidein Step D. UPLC Method B: m/e=611.202 [M+1]; Rt=1.88 min.

Example 4

The synthesis of oligosaccharide linker6-[(2,5-dioxopyrrolidin-1-yl)oxy]-N-{2-[(α-L-fucopyranosyl)oxy]ethyl}-6-oxohexanamide(ML-4) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-1 substituting 2-aminoethyl α-L-fucopyranoside(Bilstein J. Org. Chem. 2010, 6, 699-703) for 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosidein Step B. UPLC Method B: m/e=433.14 [M+1]; Rt=2.14 min.

Example 5

The synthesis of oligosaccharide linker6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-N-[2-(α-D-mannopyranosyloxy)ethyl]-6-oxohexanamide(ML-5) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-1 substituting 2-aminoethyl α-D-mannopyranoside (Eur.J. Org. Chem. 2002, 79-86) for 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosidein Step B. UPLC Method B: m/e=449.14 [M+1], Rt=1.90 min.

Example 6

The synthesis of oligosaccharide linkerN,N-Bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]-6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexanamide(ML-6) having the following structure is described.

Step A: benzyl 6-[bis(2-tert-butoxy-2-oxoethyl)amino]-6-oxohexanoate

To a stirred solution of 6-(benzyloxy)-6-oxohexanoic acid (1.5 g, 6.35mmol) in DMF (50 mL) at room temperature was added DIPEA (2.218 mL,12.70 mmol), HOBt (1.945 g, 12.7 mmol), EDC (2.434 g, 12.7 mmol) anddi-tert-butyl 2,2′-iminodiacetate (2.34 g, 9.52 mmol). After stirring atroom temperature for 16 hours, the reaction mixture was diluted with H₂O(30 mL) and extracted with CH₂Cl₂ (2×30 mL). The combined organic phasewas washed with brine, dried over Na₂SO₄ and concentrated. The residuewas purified by flash chromatography on silica gel (80 g), eluting with0-40% EtOAc in hexane, to give the title compound. LC-MS Method A:m/e=464.04 [M+1]; Rt=2.47 min. ¹H NMR (CDCl₃) δ 7.32 (m, 5H), 5.07 (s,2H), 4.02 (s, 2H), 3.96 (s, 2H), 2.35 (s, 2H), 2.26 (s, 2H), 1.66 (s,4H), 1.42-1.44 (bs, 18H).

Step B: 2,2′-{[6-(benzyloxy)-6-oxohexanoyl]imino}diacetic acid

To a stirred solution of benzyl6-[bis(2-tert-butoxy-2-oxoethyl)amino]-6-oxohexanoate (5.9 g, 12.73mmol) in CH₂Cl₂ (30 mL) at room temperature was added TFA (30 mL, 12.73mmol). After stirring at room temperature for 16 hours, the mixture wasconcentrated. The residue was purified by flash chromatography on C18reverse phase silica gel to give the title compound. LC-MS Method A:m/e=486 [M+1]; Rt=2.53 min. ¹H NMR (CD₃OD) δ 7.30 (m, 5H), 5.06 (s, 2H),4.81 (s, 4H), 4.19 (s, 2H), 4.07 (s, 2H), 2.34 (q, 4H, J=7.03).

Step C: benzyl6-{bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}-6-oxohexanoate

To a stirred solution of2,2′-{[6-(benzyloxy)-6-oxohexanoyl]imino}diacetic acid (800 mg, 2.277mmol) in DMF (12 mL) at room temperature was added 2-aminoethylα-L-fucopyranoside (1.132 g, 5.46 mmol), DMAP (834 mg, 6.83 mmol) andEDC (1.528 g, 7.97 mmol). After stirring at rt for 16 hr, the reactionmixture was concentrated and the residue was purified by flashchromatography on C18 reverse phase silica gel (120 g), eluting with0-40% AcCN in water, to give the title compound. LC-MS Method A:m/e=730.26 [M+1]; Rt=1.42 min.

Step D:6-{bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}-6-oxohexanoicacid

To a stirred solution of benzyl6-{bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}-6-oxohexanoate(900 mg, 1.233 mmol) in H₂O (5 mL) at rt was added dihydroxypalladium(866 mg, 1.233 mmol). The mixture was degassed and then stirred under aballoon of H₂. After stirring at rt under H₂ for 16 hr, the reactionmixture was filtered through a Celite pad and washed with CH₃OH (3×10mL). The filtrate was concentrated to give the title compound. LC-MSMethod A: m/e=640.17 [M+1]; Rt=0.98 min.

Step E:N,N-bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]-6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexanamide

To a stirred solution of6-{bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}-6-oxohexanoicacid (160 mg, 0.250 mmol) in DMF (3.0 mL) at 0° C. was added a solutionof TSTU (94 mg, 0.313 mmol) in DMF (2 mL) and, 5 min later, DIPEA (53μL, 0.300 mmol). After stirring for 1.5 h at 0° C., the mixture wasadded dropwise to Et₂O (30 mL) in a centrifuge tube. After centrifugedfor 30 min at 3500 rpm, the supernatant was decanted and the solidresidue was dissolved in H₂O, which was freeze-dried to give the titleproduct. UPLC Method B: m/e=737 [M+1]; Rt=2.19 min.

Example 7

The synthesis of oligosaccharide linker2,2′-{[2-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl]imino}bis(N-{2-[(α-L-fucopyranosyl)oxy]ethyl}acetamide)(ML-7) having the following structure is described.

Step A:2,2′-[(2-{[6-(benzyloxy)-6-oxohexyl]amino}-2-oxoethyl)imino]diaceticacid

To a solution of 6-(benzyloxy)-6-oxohexan-1-aminium4-methylbenzenesulfonate (2.0 g, 5.08 mmol) in DMF (10 mL) at 0° C. wasadded K₂CO₃ (738 mg, 5.34 mmol). After stirring at 0° C. for 2 hr, thesupernant of the reaction mixture was added to a solution of3-(2,6-dioxomorpholin-4-yl)propanoic acid (1.10 g, 6.35 mmol) in DMF (10mL) at 0° C. After stirring at 0° C. for 30 min, the reaction mixturewas allowed to stir at rt for 1 hr and then cooled down to 0° C.followed by the addition of water (10 mL). The resulting mixture wasconcentrated and the residue was suspended in water (10 mL). Afterstirring at 0° C. for 16 hr, the solid was collected through filtrationand dried to yield the title compound. ¹H NMR (CD₃OD) δ 7.36-7.30 (m,5H), 5.11 (s, 2H), 3.56 (s, 4H), 3.43 (s, 2H), 3.23 (t, J=6.7, 2H), 2.39(t, J=7.3, 2H), 1.68-1.62 (m, 2H), 1.57-1.51 (m, 2H), 1.40-1.35 (m, 2H).

Step B: benzyl6-[({bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}acetyl)amino]hexanoate

To a solution of 2-aminoethyl α-L-fucopyranoside (7.88 g, 38.04 mmol)and 2,2′-[(2-{[6-(benzyloxy)-6-oxohexyl]amino}-2-oxoethyl)imino]diaceticacid (2.5 g, 19.02 mmol) in DMF (10 mL) was added HOBt (2.43 g, 15.85mmol) and EDC (3.04 g, 15.85 mmol). After stirring at rt for 16 hr, thereaction mixture was concentrated and the residue was purified by flashchromatography on C18 reverse phase silica gel (120 g), eluting with0-50% AcCN in water, to give the title compound. UPLC Method B:m/e=773.292 [M+1]; Rt=3.74 min.

Step C:2,2′-{[2-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl]imino}bis(N-{2-[(α-L-fucopyranosyl)oxy]ethyl}acetamide)

The title compound was prepared using procedures analogous to thosedescribed for ML-6 substituting benzyl6-[({bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}acetyl)amino]hexanoatefor benzyl6-{bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}-6-oxohexanoatein Step D. UPLC Method B: m/e=780.265 [M+1]; Rt=2.39 min.

Example 8

The synthesis of oligosaccharide linker 2,5-Dioxopyrrolidin-1-ylN,N-bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]glycyl-β-alaninate(ML-8) having the following structure is described.

Step A:2,2′-[(2-{[3-(benzyloxy)-3-oxopropyl]amino}-2-oxoethyl)imino]diaceticacid

To an ice bath cooled solution of benzyl 3-aminopropanoate hydrochloride(4.49 g, 20.8 mmol) in DMF (30 mL) was added K₂CO₃ (3.02 g, 21.84 mmol)and the resulting mixture was stirred at o° C. for 2 hr. The mixture wasthen filtered and the filtrate was added to an ice bath cooled solutionof 2-(2,6-dioxomorpholino)acetic acid (4.47 g, 25.8 mmol) in DMF (30mL). The resulting mixture was stirred at 0° C. for 30 min, then at rtfor 2 hr. The reaction was quenched by the addition of water (30 mL) andthe resulting mixture was concentrated. The residue was stirred withwater (40 mL), and the resulting precipitate was collected throughfiltration and dried to give the title compound. ¹H NMR (DMSO-d6) δ 2.53(t, J=6.8, 2H), 3.27 (s, 2H), 3.36 (q, J=6.2, 2H), 3.42 (m, m 2H), 3.49(s, 2H), 5.09 (s, 2H), 7.28 (m, 4H), 8.16 (m, 1H), 12.45 (br s, 2H).

Step B: 2,5-Dioxopyrrolidin-1-ylN,N-bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]glycyl-β-alaninate

The title compound was prepared using procedures analogous to thosedescribed for ML-7 substituting2,2′-[(2-{[3-(benzyloxy)-3-oxopropyl]amino}-2-oxoethyl)imino]diaceticacid for2,2′-[(2-{[6-(benzyloxy)-6-oxohexyl]amino}-2-oxoethyl)imino]diaceticacid in Step B. UPLC Method E: m/e=738.2149 [M+1; Rt=1.77 min.

Example 9

The synthesis of oligosaccharide linker 2,5-Dioxopyrrolidin-1-ylN,N-bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]glycylglycinate(ML-9) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-7 substituting benzyl glycinate for6-(benzyloxy)-6-oxohexan-1-aminium 4-methylbenzenesulfonate in Step A.UPLC Method B: m/e=724.23 [M+1]; Rt=1.10 min.

Example 10

The synthesis of oligosaccharide linker15-[(2,5-Dioxopyrrolidin-1-yl)oxy]-N-{2-[(α-L-fucopyranosyl)oxy]ethyl}-3-[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]-5,15-dioxo-9,12-dioxa-3,6-diazapentadecan-1-amide(ML-10) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-7 substituting benzyl3-[2-(2-aminoethoxyl)ethoxy]propanoate for6-(benzyloxy)-6-oxohexan-1-aminium 4-methylbenzenesulfonate in Step A.UPLC Method B: m/e=825.812 [M+1]; Rt=2.17 min.

Example 11

The synthesis of oligosaccharide linker 2,5-Dioxopyrrolidin-1-yl6-{[bis({3-oxo-3-[(α-L-fucopyranosyl)oxy]-2-oxoethyl}amino)propyl]amino}-6-oxohexanoate(ML-11) having the following structure is described.

Step A: 3,3′-{[6-(benzyloxy)-6-oxohexanoyl]imino}dipropanoic acid

To a solution of 3,3′-iminodipropionic acid (2.59 g, 7.76 mmol) in DMF(20 mL) at 0° C. was added benzyl6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohenxanoate (2.59 g, 7.76 mmol) inDMF (3 mL) portionwise over a period of 15 min and then TEA (951 μL,6.83 mmol) dropwise over a period of 10 min. The resulting suspensionwas stirred at rt for 16 hr. The insoluble material was removed byfiltration and the filtrate was concentrated. The residue was purifiedby flash chromatography on C18 reverse phase silica gel (150 g), elutingwith 5-50% AcCN in water, to give the title compound. UPLC Method B:m/e=380.177 [M+1]; Rt=3.46 min.

Step B: 2,5-dioxopyrrolidin-1-yl6-{[bis({3-oxo-3-[(α-L-fucopyranosyl)oxy]-2-oxoethyl}amino)propyl]amino}-6-oxohexanoate

The title compound was prepared using procedures analogous to thosedescribed for ML-6 substituting3,3′-{[6-(benzyloxy)-6-oxohexanoyl]imino}dipropanoic acid for2,2′-{[6-(benzyloxy)-6-oxohexanoyl]imino}diacetic acid in Step C. UPLCMethod B: m/e=765.36 [M+1]; Rt=2.15 min.

Example 12

The synthesis of oligosaccharide linker1-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-1-oxohexan-2-yl]-N-{2-[(α-L-fucopyranosyl)oxy]ethyl}-N′-[(2S)-6-{[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]amino}hexanediamide(ML-12) having the following structure is described.

Step A:N²-[(benzyloxy)carbonyl]-N⁶-[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]-L-lysine

In a 250 mL round bottom flask was addedN²-[(benzyloxy)carbonyl]-L-lysine (194 mg, 0.694 mmol) and DMF (10 mL).To the above solution was added ML-4 (300 mg, 0.694 mmol) in DMF (5 mL)dropwise, followed by the addition of DIPEA (121 μL, 0.694 mmol). Afterstirring at rt for 18 hr, the reaction mixture was concentrated. Theresidue was purified by flash chromatography on C18 reverse phase silicagel eluting with 0-40% AcCN in water to give the title product. UPLCMethod B: m/e=598.2997 [M+1]; Rt=2.99 min.

Step B:N⁶-[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]-L-lysine

In a 100 mL flask,N²-[(benzyloxy)carbonyl]-N⁶-[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]-L-lysine(200 mg, 0.335 mmol) was dissolved in water (5 mL). The flask wasdegassed and filled with N₂. To the above mixture was added PdOH2 (48.7mg, 0.069 mmol. The mixture was stirred under H₂ balloon for 2 hr. Themixture was filtered through a pad of Celite, and the filtrate wasconcentrated to give the title compound. UPLC Method B: m/e=464.2697[M+1]; Rt=2.61 min.

Step C: benzyl6-({N²,N⁶-bis[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]-L-lysyl}amino)hexanoate

In a 40 mL vial was addedN⁶-[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]-L-lysine(150 mg, 0.324 mmol) and DMF (5 mL). The solution was cooled to 0° C. Tothe above solution was added ML-4 (140 mg, 0.324 mmol) in DMF (2 mL)dropwise followed by the addition of TEA (45 μL, 0.324 mmol). Thereaction mixture was warmed to rt and stirred for 1 h. To the resultingmixture was added TSTU (97 mg, 0.324 mmol) followed by the addition ofTEA (45 μL, 0.324 mmol). The mixture was stirred at rt for 20 min. Tothe resulting mixture was added a solution of6-(benzyloxy)-6-oxohexan-1-aminium 4-methylbenzenefulfonate (127 mg,0.324 mmol) in DMF (1.0 mL). After stirring at rt for 18 hr, the mixturewas concentrated. The residue was purified by flash chromatography togive the title compound. UPLC Method B: m/e=984.5469 [M+1]; Rt=3.37 min.

Step D:1-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-1-oxohexan-2-yl1-N-{2-[(α-L-fucopyranosyl)oxy]ethyl}-N′-(2S)-6-{[6-({2-[(α-L-galactopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]amino}hexanediamide

The title compound was prepared using procedures analogous to thosedescribed for ML-1 substituting benzyl6-({N²,N⁶-bis[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]-L-lysyl}amino)hexanoatefor benzyl6-({2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}amino)-6-oxohexanoatein Step C. UPLC Method B: m/e=991.5182 [M+1]; Rt=2.41 min.

Example 13

The synthesis of oligosaccharide linker 2N-{2-[(α-L-Fucopyranosyl)oxy]ethyl}-N′-(5S)-6-{[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexyl]amino}-5-({8-[(2,5-dioxopyrrolidin-1-yl)oxy]-8-oxooctanoyl}amino)-6-oxohexyl]hexanediamide(ML-13) having the following structure is described.

Step A:N²-{8-(benzyloxy)-8-oxooctanoyl}-N⁶-[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]-L-lysine

To a solution ofN⁶-[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]-L-lysine(310 mg, 0.669 mmol) in DMF (20 mL) at 0° C. was added benzyl8-[(2,5-dioxopyrrolidin-1-yl)oxy]-8-oxooctanoate (242 mg, 0.669 mmol),followed by the addition of DIPEA (0.117 ml, 0.669 mmol). The reactionwas warmed to 25° C. and stirred at this temp for 18 hr. The reactionmixture was concentrated, and the residue was purified by flashchromatography on C18 reverse phase silica gel, eluting with 0-30% CANin water, to give the title compound. UPLC Method B: m/e=710.423 [M+1];Rt=4.59 min.

Step B: benzyl8-({(12S)-1,26-bis[(α-L-fucopyranosyl)oxy]-4,11,18,23-tetraoxo-3,10,17,24-tetraazahexacosan-12-yl}amino)-8-oxooctanoate

In a 40 mL vial was addedN²-{8-(benzyloxy)-8-oxooctanoyl}-N⁶-[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]-L-lysine(100 mg, 0.141 mmol) and DMF (5 mL). To the above solution at 0° C. wasadded EDC (40.5 mg, 0.211 mmol) and HOBt (23.7 mg, 0.155 mmol). Thereaction was warmed to rt and stirred at rt for 20 min. To the abovemixture was added 6-amino-N-{2-[(α-L-fucopyranosyl)oxy]ethyl}hexanamide(45.1 mg, 0.141 mmol). After stirring at rt for 18 hr, the mixture wasconcentrated. The residue was purified by flash chromatography elutingwith 0-40% AcCN in water to give the title compound. UPLC Method B:m/e=1012.6348 [M+1]; Rt=3.28 min.

Step C:N-{2-[(α-L-fucopyranosyl)oxy]ethyl}-N′-[(5S)-6-{[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexyl]amino}-5-({8-[(2,5-dioxopyrrolidin-1-yl)oxy]-8-oxooctanoyl}amino)-6-oxohexyl]hexanediamide

The title compound was prepared using procedures analogous to thosedescribed for ML-1 substituting benzyl8-({(12S)-1,26-bis[(α-L-fucopyranosyl)oxy]-4,11,18,23-tetraoxo-3,10,17,24-tetraazahexacosan-12-yl}amino)-8-oxooctanoatefor benzyl6-({2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}amino)-6-oxohexanoatein Step C. UPLC Method B: m/e=1019.588 [M+1]; Rt=2.38 min.

Example 14

The synthesis of oligosaccharide linkerN-{(5S)-5-({8-[(2,5-Dioxopyrrolidin-1-yl)oxy]-8-oxooctanoyl}amino)-6-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-6-oxohexyl}-N′-{2-[(α-L-fucopyranosyl)oxy]ethyl}hexanediamide (ML-14) having the following structure is described.

Step A: 2,5-dioxopyrrolidin-1-ylN²-[(benzyloxy)carbonyl]-N⁶-[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]-L-lysinate

The title compound was prepared using the procedure analogous to thatdescribed for ML-1 Step A, substitutingN²-{8-(benzyloxy)-8-oxooctanoyl}-N⁶-[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]-L-lysinefor 6-(benzyloxy)-6-oxohexanoate. UPLC Method B: m/e=695.213 [M+1];Rt=3.98 min.

Step B:N²-[(benzyloxy)carbonyl]-N⁶-[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)-L-lysinamide

In a 40 mL vial, 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranoside(883 mg, 1.612 mmol) was dissolved in DMF (10 mL). To the above solutionat 0° C. was added a solution of 2,5-dioxopyrrolidin-1-ylN²-[(benzyloxy)carbonyl]-N⁶-[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]-L-lysinate(700 mg, 1.008 mmol) in DMF (10 mL) dropwise. After stirring at rt for18 hr, the mixture was concentrated. The residue was purified by HPLC(waters Delta Pak C4 300 A, 15 um, 50×250 mm column, flow rate 85ml/min, gradient 8-30% ACN/water in 25 min) to give the title compound.UPLC Method B: m/e=1127.335 [M+1]; Rt=2.83 min.

Step C:N-{(5S)-5-amino-6-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-6-oxohexyl}-N′-{2-[(α-L-fucopyranosyl)oxy]ethyl}hexanediamide

In a 100 mL flask,N²-[(benzyloxy)carbonyl]-N⁶-[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)-L-lysinamide(950 mg, 0.843 mmol) was dissolved in water (10 mL). The flask wasdegassed and filled with N₂. To the resulting mixture was added Pd/C(10%, 179 mg, 0.169 mmol). The mixture was stirred under H₂ balloon for18 hr. The mixture was filtered through a pad of Celite, and thefiltrate was concentrated to give the title compound. UPLC Method B:m/e=993.326 [M+1]; Rt=1.37 min.

Step D:N-{(5S)-5-({8-[(2,5-dioxopyrrolidin-1-yl)oxy]-8-oxooctanoyl}amino)-6-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-6-oxohexyl}-N′-{2-[(α-L-fucopyranosyl)oxy]ethyl}hexanediamide

The title compound was prepared using procedure analogous to thosedescribed for ML-12 substitutingN-{(5S)-5-amino-6-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-6-oxohexyl}-N′-{2-[(α-L-fucopyranosyl)oxy]ethyl}hexanediamideforN⁶-[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]-L-lysinein Step C: UPLC Method B: m/e=1218.418 [M+1]; Rt=2.25 min.

Example 15

The synthesis of oligosaccharide linker2,2′-{[2-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl]imino}bis[N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)acetamide](ML-15) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-7 substituting 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosidefor 2-aminoethyl α-L-fucopyranoside in Step B. UPLC Method B:m/e=1460.58 [M+1]; Rt=1.53 min.

Example 16

The synthesis of oligosaccharide linker N,N-Bis{2-[(α-L-fucopyranosyl)oxy]ethyl}-1-{6-[2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexanoyl}pyrrolidine-cis-3,4-dicarboxamide(ML-16) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-6 substituting pyrrolidine-cis-3,4-dicarboxylic acidfor 2,2′-{[6-(benzyloxy)-6-oxohexanoyl]imino}diacetic acid in Step C.UPLC Method B: m/e=763.38 [M+1]; Rt=2.12 min.

Example 17

The synthesis of oligosaccharide linker1-{6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexanoyl}-N,N′-bis{2-[(α-L-fucopyranosyl)oxy]ethyl}piperidine-cis-3,5-dicarboxamide(ML-17) having the following structure is described.

Step A:N,N′-bis{2-[(α-L-fucopyranosyl)oxy]ethyl}pyridine-3,5-dicarboxamide

To a stirred solution of 3,5-pyridinedicarboxylic acid (311 mg, 1.861mmol) in DMF (30 mL) at room temperature was added 2-aminoethylα-L-fucopyranoside (2.077 g, 9.30 mmol), DMAP (568 mg, 4.65 mmol), andEDC (1784 mg, 9.30 mmol). After stirring at room temperature for 16hours, the reaction mixture was concentrated. The residue was purifiedby flash chromatography on C18 reverse phase silica gel (300 g), elutingwith AcCN in water) to give the title compound. UPLC Method B:m/e=578.31 [M+1]; Rt=0.25 min.

Step B:N,N′-bis{2-[(α-L-fucopyranosyl)oxy]ethyl}piperidine-cis-3,5-dicarboxamide

To a stirred solution of N,N′-bis{2-[(α-L-fucopyranosyl)oxy]ethyl}pyridine-3,5-dicarboxamide (550 mg,0.952 mmol) in H₂O (12 mL) at room temperature was added PtO₂ (64.9 mg,0.286 mmol). The mixture was degassed and then stirred under a balloonof H₂ at rt for 4 hr. The reaction mixture was then filtered through aCelite pad, and the filtrate was concentrated and redissolved in CH₃OH,centrifuged to precipitate the solid catalyst. The supernatant wasconcentrated to give the title compound. UPLC Method B: m/e=584.27[M+1]; Rt=1.14 min.

Step C: benzyl6-[cis-3,5-bis({2-[(α-L-fucopyranosyl)oxy]ethyl}carbamoyl)piperidin-1-yl]-6-oxohexanoate

To a stirred solution of 6-(benzyloxy)-6-oxohexanoic acid (125 mg, 0.529mmol) in DMF (3 mL) at room temperature was added DMAP (64.6 mg, 0.529mmol), EDC (203 mg, 1.058 mmol) and a solution ofN,N′-bis{2-[(α-L-fucopyranosyl)oxy]ethyl}piperidine-cis-3,5-dicarboxamide(438 mg, 0.794 mmol) in DMF (2 mL). After stirring at rt for overnight,the reaction mixture was concentrated and the residue was purified byflash chromatography on C18 reverse phase silica gel, eluting with 0-50%AcCN in water, to afford the title compound. UPLC Method F: m/e=770.48[M+1]; Rt=1.38 min.

Step D:1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexanoyl}-N,N′-bis{2-[(α-L-fucopyranosyl)oxy]ethyl}piperidine-cis-3,5-dicarboxamide

The title compound was prepared using procedures analogous to thosedescribed for ML-6 substituting benzyl6-[cis-3,5-bis({2-[(α-L-fucopyranosyl)oxy]ethyl}carbamoyl)piperidin-1-yl]-6-oxohexanoatefor benzyl6-{bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}-6-oxohexanoatein Step D. UPLC Method F: m/e=777.35 [M+1]; Rt=2.23 min.

Example 18

The synthesis of oligosaccharide linkerN¹,N⁵-Bis{2-[(α-L-fucopyranosyl)oxy]ethyl}-N²-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexanoyl}-L-glutamamide(ML-18) having the following structure is described.

Step A: benzyl[(2S)-1,5-dioxin-1,5-bus({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)pentane-2-yl]carbamate

To a solution of N-[(benzyl)carbonyl]-L-glutei acid (1.1 g, 3.91 mmol)and 2-amino ethyl α-L-fucopyranoside (2.026 g, 9.78 mmol) in DMF (10 mL)was added EDC (3.00 g, 15.64 mmol) and DMAP (0.048 g, 0.391 mmol). Afterstirring at rt for 24 hr, the reaction mixture was concentrated and theresidue was purified by flash chromatography on silica gel (80 g),eluting with 100% EtOAc for 5 column volume and then isocraticEtOAc:AcCN:MeOH 6:1:1 to give the title compound. ¹H NMR (CD₃OD) δ7.38-7.29 (m, 5H), 5.13-5.05 (m, 2H), 4.76 (s, 2H), 4.09 (dd, J=5.3,8.6, 1H), 3.95-3.91 (m, 2H), 3.74-3.65 (m, 6H), 3.53-3.25 (m, 8H), 2.30(t, J=7.5, 2H), 2.06-2.04 (m, 1H), 1.92-1.89 (m, 1H), 1.19 (d, J=6.5,6H).

Step B:2-[(N-{2-[(α-L-fucopyranosyl)oxy]ethyl}-L-α-glutaminyl)amino]ethylα-L-fucopyranoside

A suspension of benzyl[(2S)-1,5-dioxo-1,5-bis({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)pentan-2-yl]carbamate(940 mg, 1.425 mmol) and Pearlman's catalyst (20 mg, 0.028 mmol) inCH₃OH (20 mL) was shaked under 30 Psi of H₂ at room temperature. After16 hours, the catalyst was filtered off and the filtrate wasconcentrated to the title compound.

Step C: benzyl{[(2S)-1,5-dioxo-1,5-bis({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)pentan-2-yl]amino}-6-oxohexanoate

To a solution of 6-(benzyloxy)-6-oxohexanoic acid (260 mg, 1.1 mmol) inDMF (10 mL) at 0° C. was added TSTU (348 mg, 1.155 mmol) followed byDIPEA (202 μL, 1.155 mmol). After stirring at 0° C. for 1 hr, thereaction mixture was partitioned between Et₂O (100 mL) and brine (100mL). The organic phase was separated, further washed with brine (2×100mL), dried over MgSO₄, and concentrated. The residue was redissolved inDMF (5 mL), added to a solution of2-[(N-{2-[(α-L-fucopyranosyl)oxy]ethyl}-L-α-glutaminyl)amino]ethylα-L-fucopyranoside (368 mg, 1.103 mmol) in DMF (10 mL) at 0° C. followedby adding Et₃N (154 μL, 1.103 mmol). After stirring at 0° C. for 30 min,the reaction mixture was allowed to gradually warm up to rt and stir for2 h. The reaction mixture was diluted with CH₃OH (10 mL) and purified byHPLC (gradient 6-30% 0.1% TFA in water over 34 min, 50×250 mm C4 15 um,300 A, 100 mL/min flow rate). ¹H NMR (CD₃OD) δ 8.18 (m, 1H), 7.35-7.30(m, 5H), 5.11 (s, 2H), 4.76 (d, J=3.6, 2H), 4.30 (dd, J=5.5, 8.6, 1H),3.93 (dd, J=6.5, 13.1, 2H), 3.74-3.71 (m, 4H), 3.65 (s, 2H), 3.54-3.25(m, 6H), 2.40 (t, J=6.7, 2H), 2.29-2.26 (m, 4H), 2.07-2.03 (m, 2H),1.93-1.88 (m, 2H), 1.65-1.64 (m, 4H), 1.19 (d, J=6.5, 6H).

Step D:{[(2S)-1,5-dioxo-1,5-bis({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)pentan-2-yl]amino}-6-oxohexanoicacid

A suspension of benzyl{[(2S)-1,5-dioxo-1,5-bis({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)pentan-2-yl]amino}-6-oxohexanoate(380 mg, 0.511 mmol) and Pearlman's catalyst (50 mg, 0.071 mmol) inmethanol (30 mL) was agitated under 50 Psi of H₂ at room temperature ona Parr shaker. After 16 hours, catalyst was filtered off and thefiltrate was concentrated to give the title compound. ¹H NMR (CD₃OD) δ4.76-4.75 (m, 2H), 4.32-4.29 (m, 1H), 3.96-3.91 (m, 2H), 3.76-3.66 (m,5H), 3.55-3.27 (m, 9H), 2.34-2.27 (m, 6H), 2.09-2.04 (m, 1H), 1.99-1.88(m, 1H), 1.67-1.61 (m, 4H), 1.20 (d, J=6.7, 6H).

Step E:N¹,N⁵-bis{2-[(α-L-fucopyranosyl)oxy]ethyl}-N²-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexanoyl}-L-glutamamide

A suspension of{[(2S)-1,5-dioxo-1,5-bis({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)pentan-2-yl]amino}-6-oxohexanoicacid (289 mg, 0.422 mmol) in DMF (5.0 mL) at 0° C. was added TSTU (140mg, 0.464 mmol) followed by Hunig's base (810 μL, 0.464 mmol). Afterstirring for 1 hour, the mixture was concentrated to give the titleproduct, which was used without further purification. UPLC Method B:m/e=[M+1]; Rt=1.82 min.

Example 19

The synthesis of oligosaccharide linker N¹,N⁴-Bis{2-[(α-L-fucopyranosyl)oxy]ethyl}-N²-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexanoyl}-L-aspartamide(ML-19) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-18 substituting N-[(benzyloxy)carbonyl]-L-aspartic acidfor N-[(benzyloxy)carbonyl]-L-glutamic acid in Step A. UPLC Method B:m/e=737.3126 [M+1]; Rt=2.04 min.

Example 20

The synthesis of oligosaccharide linkerN²-{6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexanoyl}-N⁵-{2-[(α-L-fucopyranosyl)oxy]ethyl}-N′-{2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}-L-glutamamide(ML-20) having the following structure is described.

Step A: benzylN²-[(benzyloxy)carbonyl]-N-{2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}-L-glutaminate

To a solution of Z-Glu-γ-Bn (1.0 g, 2.69 mmol) and 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranoside(2.21 g, 4.04 mmol) in DMF (10 mL) was added EDC (1.29 g, 6.73 mmol),HOBt (41 mg, 0.269 mmol), and Et₃N (38 μL, 0.269 mmol). After stirringat room temperature for 16 hours, the mixture was purified by HPLC(50×250 mm, C4, flow rate 85 mL/minutes, gradient 25-35% AcCN in H₂Owith 0.1% TFA over 30 min) to give the title compound. ¹H NMR (CD3OD) δ8.12-8.10 (m, 1H), 7.38-7.26 (m, 10H), 5.50-5.04 (m, 5H), 4.81 (s, 1H),4.73 (s, 1H), 4.16-4.13 (m, 1H), 4.06 (s, 1H), 3.99-3.3.97 (m, 1H),3.93-3.37 (m, 20H), 2.48 (t, J=7.6, 2H), 2.15-2.10 (m, 1H), 1.97-1.90(m, 1H).

Step B:N-{2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}-L-glutamine

A mixture of benzylN²-[(benzyloxy)carbonyl]-N-{2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}-L-glutaminate(1.41 g, 1.57 mmol) and Pearlman's catalyst (110 mg, 0.157 mmol) in H₂O(30 mL) was agitated under 50 Psi H₂ on a Parr shaker at roomtemperature. After 16 hours, catalyst was filtered off and the filtratewas freeze-dried to give the title compound. ¹H NMR (D₂O) δ 5.07 (s,1H), 4.87 (s, 1H), 4.81 (s, 1H), 4.08-3.55 (m, 22H), 3.41-3.36 (m, 1H),2.32 (t, J=7.5, 2H), 2.10-2.06 (m, 2H).

Step C: N²-[6-(benzyloxy)-6-oxohexanoyl1-N-{2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}-L-glutamine

The title compound was prepared using procedure analogous to thatdescribed for ML-18 substitutingN-{2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}-L-glutaminefor 2-[(N-{2-[(α-L-fucopyranosyl)oxy]ethyl}-L-α-glutaminyl)amino]ethylα-L-fucopyranoside in Step C. ¹H NMR (CD₃OD) δ 8.09-8.06 (m, 1H),7.35-7.30 (m, 5H), 5.11 (s, 2H), 5.08 (s, 1H), 4.79 (m, 1H), 4.72 (s,1H), 4.34-4.31 (m, 1H), 4.06-3.37 (m, 22H), 2.42-2.36 (m, 4H), 2.29-2.26(m, 2H), 2.09-2.05 (m, 1H), 1.93-1.88 (m, 1H), 1.65-1.62 (m, 4H).

Step D:N²-[6-(benzyloxy)-6-oxohexanoyl]-N⁵-{2-[(α-L-fucopyranosyl)oxy]ethyl}-N¹-{2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}-L-glutamamide

To a solution ofN²-[6-(benzyloxy)-6-oxohexanoyl]-N-{2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}-L-glutamine(500 mg, 0.559 mmol) and 2-aminoethyl α-L-fucopyranoside (116 mg, 0.559mmol) in DMF (10 mL) was added EDC (161 mg, 0.838 mmol) and HOBt (8.56mg, 0.056 mmol). After stirring at room temperature for 16 hours, themixture was purified by HPLC (50×250 mm, C4, flow rate 85 mL/minutes,gradient 25-35% AcCN in H₂O with 0.1% TFA over 30 min) to give the titlecompound. ¹H NMR (CD3OD) δ 8.12-8.08 (m, 1H), 7.35-7.29 (m, 5H), 5.11(s, 2H), 5.08 (s, 1H), 4.80 (s, 1H), 4.77 (s, 1H), 4.72 (s, 1H),4.32-4.29 (m, 1H), 4.12-3.26 (m, 30H), 2.42-2.39 (m, 2H), 2.30-2.26 (m,4H), 2.09-2.04 (m, 1H), 1.93-1.88 (m, 1H), 1.65-1.64 (m, 4H), 1.19 (d,J=6.7, 3H).

Step E:N²-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexanoyl}-N⁵-{2-[(α-L-fucopyranosyl)oxy]ethyl}-N¹-{2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}-L-glutamamide

The title compound was prepared using procedures analogous to thosedescribed for ML-18 substitutingN²-[6-(benzyloxy)-6-oxohexanoyl]-N⁵-{2-[(α-L-fucopyranosyl)oxy]ethyl}-N¹-{2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}-L-glutamamidefor benzyl{[(2S)-1,5-dioxo-1,5-bis({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)pentan-2-yl]amino}-6-oxohexanoatein Step D. ¹H NMR (CD₃OD) δ 5.08 (s, 1H), 4.80 (s, 1H), 4.77 (s, 1H),4.72 (s, 1H), 4.33-4.30 (m, 1H), 4.06-3.33 (m, 30H), 2.84-2.82 (m, 4H),2.69-2.66 (m, 2H), 2.34-2.27 (m, 4H), 2.10-2.02 (m, 1H), 1.94-1.89 (m,1H), 1.76-1.74 (m, 4H), 1.20 (d, J=6.5, 3H).

Example 21

The synthesis of oligosaccharide linker 2,5-Dioxopyrrolidin-1-ylN²-[5-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-5-oxopentanoyl]-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)-L-glutaminylglycinate(ML-21) having the following structure is described.

Step A: (S)-benzyl2-{[(benzyloxy)carbonyl]amino}-5-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-5-oxopentanoate

To a solution of 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranoside(2.6 g, 4.75 mmol), and(S)-5-(benzyloxy)-4-{[(benzyloxy)carbonyl]amino}-5-oxopentanoic acid(2.0 g, 5.39 mmol) in DMF (36 mL) at 0° C. was added DMAP (580 mg, 4.75mmol) and EDC (3.64 g, 19.00 mmol). The reaction mixture was allowed togradually warm up to rt. After stirring for 16 hr, the reaction mixturewas concentrated and the residue was purified by flash chromatography onC18 reverse phase silica gel (275 g), eluting with 10-55% AcCN in H₂O togive the title compound. UPLC Method B: calculated for C₄₀H₅₆N₂O₂₁900.34, observed m/e: 901.26 [M+1]; Rt=2.46 min.

Step B:(S)-2-amino-5-((2-((α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy)ethyl)amino)-5-oxopentanoicacid

A mixture of (S)-benzyl2-{[(benzyloxy)carbonyl]amino}-5-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-5-oxopentanoate(1.0 g, 1.11 mmol) and Pd/C (118 mg, 0.111 mmol) in water (10 mL) wasallowed to stir under a balloon of H₂ at room temperature for 16 hours.The catalyst was filtered off and washed with H₂O (3×10 mL). Thefiltrate was concentrated to give the title compound. UPLC Method B:calculated for C₂₅H₄₄N₂O₁₉ 676.25, observed m/e: 677.21 [M+1]; Rt=0.86min.

Step C: 2,5-dioxopyrrolidin-1-yl5-oxo-5-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)pentanoate

The title compound was prepared using the procedure analogous to thatdescribed for ML-4 substituting benzyl5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoate for benzyl6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexanoate. UPLC Method B:calculated for C₁₇H₂₆N₂O₁₀ 418.16, observed m/e: 419.11 [M+1]; Rt=2.00min.

Step D:(S)-5-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-5-oxo-2-[5-oxo-5-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)pentanamido]pentanoicacid

To a solution of(S)-2-amino-5-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-5-oxopentanoicacid (350 mg, 4.75 mmol) in DMF (5 mL) at 0° C. was added2,5-dioxopyrrolidin-1-yl5-oxo-5-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)pentanoate (216 mg,0.517 mmol, prepared according to Example 4, ML-4, Step A, substituting5-(benzyloxy)-5-oxopentanoic acid for 6-(benzyloxy)-6-oxohexanoic acidand TEA (0.2 mL, 1.435 mmol). After stirring at 0° C. for 2 hr, thereaction mixture was concentrated and the residue was purified by flashchromatography on C18 silica gel (150 g), eluting with 5-40% AcCN in H₂Oto give the title compound. UPLC Method B: calculated for C₃₈H₆₅N₃O₂₆979.39, observed m/e: 980.31 [M+1]; Rt=0.92 min.

Step E: (S)-2,5-dioxopyrrolidin-1-yl5-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-5-oxo-2-[5-oxo-5-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)pentanamido]pentanoate

To a solution of(S)-5-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-5-oxo-2-[5-oxo-5-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)pentanamido]pentanoicacid (366 mg, 0.373 mmol) in DMF (2 mL) at 0° C. was added TSTU (115 mg,0.381 mmol) and DIPEA (0.1 mL, 0.573 mmol). After stirring at 0° C. for1 hour, the reaction was quenched with TFA (60 μL, 0.784 mmol). Thereaction mixture was transferred dropwise, via autopipette, to a tubecontaining AcCN (45 mL). The resulting white suspension was centrifuged(3000 rpm, 15 minutes, at 4° C.) to generate a clear supernatant and awhite pellet. The supernatant was discarded and the white pellet waswashed with AcCN (1 mL) and dried to yield title compound UPLC Method B:calculated for C₄₂H₆₈N₄O₂₈ 1076.40, observed m/e: 1077.28 [M+1]; Rt=0.90min.

Step F: benzylN²-[5-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-5-oxopentanoyl]-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)-L-glutaminylglycinate

To a solution of (S)-2,5-dioxopyrrolidin-1-yl5-[(2-{[α-D-mannopyranosyl-(1→3[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-5-oxo-2-[5-oxo-5-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)pentanamido]pentanoate(0.35 g, 0.325 mmol) in DMF (4 mL) at 0° C. was added2-(benzyloxy)-2-oxoethanaminium chloride (77 mg, 0.382 mmol) and TEA(0.15 mL, 1.076 mmol). After stirring at rt for 24 hr, the reactionmixture was concentrated and the residue was purified by flashchromatography on C18 reverse phase silica gel (150 g), eluting with5-40% AcCN in H₂O to give the title compound. UPLC Method B: calculatedfor C₄₇H₇₄N₄O₂₇ 1126.45, observed m/e: 1127.39 [M+1]; Rt=2.84 min.

Step G: 2,5-dioxopyrrolidin-1-ylN²-[5-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-5-oxopentanoyl]-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)-L-glutaminylglycinate

The title compound was prepared using procedures analogous to thosedescribed for ML-1 substituting benzylN²-[5-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-5-oxopentanoyl]-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)-L-glutaminylglycinatefor benzyl6-({2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}amino)-6-oxohexanoatein Step C. UPLC Method B: calculated for C₄₄H₇₁N₅O₂₉ 1133.42, observedm/e: 1134.34 [M+1]; Rt=2.17 min.

Example 22

The synthesis of oligosaccharide linker2-{(2S)-8-[(2,5-Dioxopyrrolidin-1-yl)oxy]-1-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-1,8-dioxooctan-2-yl}-N′-{2-[(α-L-fucopyranosyl)oxy]ethyl}hexanediamide(ML-22) having the following structure is described.

Step A:(2S)-8-(benzyloxy)-2-{[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]amino}-8-oxooctanoicacid

To a solution of ML-4 (300 mg, 0.694 mmol) in DMF at 0° C. was added(S)-2-amino-8-(benzyloxy)-8-oxooctanoic acid (194 mg, 0.694 mmol)followed by DIPEA (121 μL, 0.694 mmol). The reaction mixture was allowedto stir at rt for 2 hr and then concentrated. The residue was purifiedby flash chromatography on C18 reverse phase silica gel, eluting with5-28% AcCN in H₂O to give the title compound. UPLC Method B: m/e=597.226[M+1]; Rt=3.45 min.

Step B:N-{(2S)-8-[(2,5-Dioxopyrrolidin-1-yl)oxy]-1-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-1,8-dioxooctan-2-yl}-N′-{2-[(α-L-fucopyranosyl)oxy]ethyl}hexanediamide

The title compound was prepared using procedures analogous to thosedescribed for ML-1 substituting(2S)-8-(benzyloxy)-2-{[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoyl]amino}-8-oxooctanoicacid for benzyl 6-(benzyloxy)-6-oxohexanoic acid in Step A. UPLC MethodB: m/e: 1133.312 [M+1]; Rt=2.23 min.

Example 23

The synthesis of oligosaccharide linker 2,5-Dioxopyrrolidin-1-yl1-[(α-L-fucopyranosyl)oxy]-13-{2-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-2-oxoethyl}-4,11,15-trioxo-3,10,13,16-tetraazadocosan-22-oate(ML-23) having the following structure is described.

Step A: benzyl(6-{2-[(α-L-fucopyranosyl)ethyl]amino}-6-oxohexyl)carbamate

To a solution of 2-aminoethyl α-L-fucopyranoside (3.0 g, 14.48 mmol) inDMF (80 mL) at rt was added 2,5-dioxopyrrolidin-1-yl6-{[(benzyloxy)carbonyl]amino}hexanoate (6.3 g, 17.37 mmol) and, 1 hrlater, TEA (4.44 mL, 31.8 mmol). After stirring for 16 h, the reactionmixture was concentrated and the residue was purified by flashchromatography on C18 reverse phase silica gel (230 g), eluting with5-40% AcCN in water to yield title compound. UPLC Method B: m/e=455.2568[M+1]; Rt=2.86 min.

Step B: 6-amino-N-{2-[(α-L-fucopyranosyl)oxy]ethyl}hexanamide

To a solution of benzyl(6-{2-[(α-L-fucopyranosyl)ethyl]amino}-6-oxohexyl)carbamate (1.72 g,3.79 mmol) in H₂O (20 mL) was added Pd/C (23 mg, 0.217 mmol). Themixture was degassed and stirred under a balloon of H₂. After 2 h, thereaction mixture was filtered through a Celite pad and the filtrate wasfreeze-dried to produce the title compound. ¹H NMR (CD₃OD) δ 1.21 (d, 3H), 1.40-1.38 (m, 2 H), 1.62-1.60 (m, 4 H), 2.23 (t, 2 H), 2.76 (t, 2H), 3.28-3.27 (m, 1 H), 3.44-3.43 (m, 1 H), 3.54-3.52 (m, 1 H), 3.66 (s,1 H), 3.75-3.74 (m, 2 H), 3.94-3.93 (m, 1 H), 4.76 (d, 1 H). UPLC MethodB: m/e=321.2323 [M+1]; Rt=3.02 min.

Step C:[(2-{[6-(benzyloxy)-6-oxohexyl]amino}-2-oxoethyl)(2-{[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexyl]amino}-2-oxoethyl)amino]aceticacid

To a suspension of2,2′-[(2-{[6-(benzyloxy)-6-oxohexyl]amino}-2-oxoethyl)azanediyl]diaceticacid (1.0 g, 2.54 mmol) in CH₂Cl₂ (30 mL) at 0° C. was addedtrifluoroacetic anhydride (448 μL, 3.17 mmol). After stirring at 0° C.for 3 hr, the mixture was cooled to −30° C., to which a solution of Et₃N(848 μL, 6.08 mmol) in DMF (20 mL) was added dropwise over 30 mins Afterstirring at −30° C. for 30 min, a mixture of6-amino-N-{2-[(α-L-fucopyranosyl)oxy]ethyl}hexanamide (812 mg, 2.54mmol) in DMF (30 mL) was added and the resulting mixture was allowed tostir at rt. After stirring for 16 hr, the mixture was concentrated andthe residue was purified by flash chromatography on C18 reverse phasesilica gel (42 g), eluting with 0-40% AcCN in water to produce the titlecompound. UPLC Method B: m/e=697.3876 [M+1]; Rt=3.398 min. ¹H NMR(CD₃OD) δ 1.23 (3 H, d, J=6.59), 1.39-1.36 (4 H, m), 1.56 (6 H, s), 1.67(6 H, d, J=10.32), 2.23 (2 H, t, J=7.50), 2.41 (2 H, t, J=7.37), 3.24 (6H, m), 3.42 (4 H, s), 3.49 (2 H, s), 3.57-3.52 (3 H, m), 3.68 (1 H, s),3.77 (3 H, t, J=1.65), 3.98-3.94 (1 H, m), 4.77 (1 H, s), 5.14 (2 H, s),7.38 (5 H, d, J=4.43).

Step D: benzyl1-[(α-L-fucopyranosyl)oxy]-13-{2-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-2-oxoethyl}-4,11,15-trioxo-3,10,13,16-tetraazadocosan-22-oate

To a solution of[(2-{[6-(benzyloxy)-6-oxohexyl]amino}-2-oxoethyl)(2-{[6-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexyl]amino}-2-oxoethyl)amino]aceticacid (800 mg, 1.148 mmol) in DMF (15 mL) was added 2-aminoethylα-D-mannopyranosyl-(1→3)]-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranoside(1.89 g, 3.44 mmol), HOBt (17.6 mg, 0.115 mmol), and EDC (770 mg, 4.02mmol). After stirring for 16 h at rt, the reaction mixture wasconcentrated and the residue was purified by flash chromatography on C18reverse phase silica gel (50 g), eluting with 10-40% AcCN in water togive the title compound. UPLC Method B: m/e=1226.5990 [M+1]; Rt=2.96min. ¹H NMR (CD₃OD) δ 1.23 (3 H, d, J=6.58), 1.38 (6 H, s), 1.56 (6 H,s), 1.69-1.65 (6 H, m), 2.23 (2 H, t, J=7.44), 2.41 (2 H, t, J=7.35),3.27-3.23 (6 H, m), 3.37 (1 H, s), 3.37 (1 H, s), 3.38 (1 H, s), 3.45 (1H, s), 3.47 (1 H, s), 3.47 (1 H, s), 3.54 (3 H, s), 3.60 (1 H, s), 3.62(2 H, s), 3.64 (1 H, s), 3.65 (1 H, s), 3.67 (2 H, s), 3.68 (3 H, s),3.88-3.72 (20 H, m), 4.07 (1 H, s), 4.76 (1 H, s), 4.78 (1 H, s), 4.84(1 H, d, J=1.69), 5.10 (1 H, s), 5.14 (2 H, s), 7.38 (5 H, d, J=4.37).

Step E: 2,5-dioxopyrrolidin-1-yl13-(2-((2-((((α-D-mannopyranosyl-(1→3)]-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl-oxy-(1-O→2))-ethylamino)-2-oxoethyl)-4,11,15-trioxo-1-(((2-(α-L-fucopyranosyl-oxy)-(1-O→2)))oxy)-3,10,13,16-tetraazadocosan-22-oate

The title compound was prepared using procedures analogous to thosedescribed for ML-6 substituting benzyl1-[(α-L-fucopyranosyl)oxy]-13-{2-[(2-{[α-D-mannopyranosyl-(1→3[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-2-oxoethyl}-4,11,15-trioxo-3,10,13,16-tetraazadocosan-22-oatefor benzyl6-{bis[2α-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}-6-oxohexanoatein Step D. UPLC Method B: m/e=1233.6006 [M+1]; Rt=2.223 min.

Example 21

The synthesis of oligosaccharide linker 2,5-Dioxopyrrolidin-1-ylN-(2-{[6-({2-[(6-deoxy-α-L-galactopyranosyl)oxy]ethyl}amino)-6-oxohexyl]amino}-2-oxoethyl)-N-[2-({6-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-6-oxohexyl}amino)-2-oxoethyl]glycyl-β-alaninate(ML-21) having the following structure is described.

Step A: benzyl{6-[(2-{[α-D-mannopyranoyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-6-oxohexyl}carbamate

To a stirred solution of 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranoside(1.8 g, 3.29 mmol) in DMF (50 mL) at 0° C. was added benzyl{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-hexyl}carbamate (1.787 g, 4.93mmol) and 30 minutes later Et₃N (1.146 mL, 8.22 mmol). After stirringfor 16 h, the reaction mixture was concentrated and the resultingresidue was purified by flash chromatography on C18 reverse phase silicagel (240 g), eluting 5-40% AcCN in water to yield the title compound. ¹HNMR (CD₃OD) δ 1.35 (br s, 2 H), 1.52 (br s, 2 H), 1.63 (br s, 2 H), 2.21(s, 2 H), 3.12 (s, 2 H), 3.37 (s, 1 H), 3.51-3.37 (br m, 5 H), 3.81-3.69(br m, 14 H), 3.98 (s, 1 H), 4.06 (s, 1 H), 4.72 (s, 1 H), 4.81 (s, 2H), 5.07 (s, 2 H), 7.35 (s, 5 H). UPLC Method B: m/e=795.303 [M+1];Rt=2.49 min.

Step B:6-amino-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)hexanamide

The title compound was prepared using procedure analogous to thosedescribed for ML-23 substituting benzyl{6-[(2-{[α-D-mannopyranoyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-6-oxohexyl}carbamatefor benzyl (6-{2-[(α-L-fucopyranosyl)ethyl]amino}-6-oxohexyl)carbamatein Step B. ¹H NMR (CD₃OD) δ 1.40 (2 H, d, J=7.97), 1.63 (4 H, d,J=12.78), 2.23 (2 H, t, J=7.37), 2.82 (2 H, q, J=8.46), 3.44-3.37 (2 H,m), 3.53-3.46 (1 H, m), 3.63-3.61 (4 H, m), 3.72-3.70 (6 H, m), 3.80 (5H, dd, J=9.96, 4.52), 3.83 (2 H, s), 3.90 (1 H, dd, J=11.05, 5.87), 3.97(1 H, s), 4.03 (1H, s), 4.72 (1 H, s), 4.81 (1 H, s), 5.06 (1 H, s).UPLC Method B: m/e 661.3543 [M+1]; Rt=3.89 min.

Step C: 2,5-dioxopyrrolidin-1-ylN-(2-{[6-({2-[(6-deoxy-α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexyl]amino}-2-oxoethyl)-N-[2-({6-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-6-oxohexyl}amino)-2-oxoethyl]glycyl-β-alaninate

The title compound was prepared using procedure analogous to thosedescribed for ML-23 substituting6-amino-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)hexanamideand benzyl N,N-bis(carboxymethyl)glycyl-3-alaninate for 2-aminoethylα-L-fucopyranoside and2,2′-[(2-{[6-(benzyloxy)-6-oxohexyl]amino}-2-oxoethyl)imino]diaceticacid, respectively in Step C, and6-amino-N-{2-[(6-deoxy-α-L-galactopyranosyl)oxy]ethyl}hexanamide for6-amino-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)hexanamidein Step D. UPLC Method E: m/e=1304.444 [M+1]; Rt=1.76 min.

Example 25

The synthesis of oligosaccharide linker 2,5-Dioxopyrrolidin-1-yl1-[(α-L-fucopyranosyl)oxy]-13-[2-({6-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-6-oxohexyl}amino)-2-oxoethyl]-4,11,15-trioxo-3,10,13,16-tetraazadocosan-22-oate(ML-25) having the following structure is described.

The title compound was prepared using procedureS analogous to thosedescribed for ML-23 substituting2,2′-[(2-{[6-(benzyloxy)-6-oxohexyl]amino}-2-oxoethyl)imino]diaceticacid for benzyl N,N-bis(carboxymethyl)glycyl-β-alaninate. UPLC Method E:m/e=1346.5950 [M+1]; Rt=2.29 min.

Example 26

The synthesis of oligosaccharide linker 2,5-Dioxopyrrolidin-1-yl1-[(α-L-fucopyranosyl)oxy]-11-[2-({4-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-4-oxobutyl}amino)-2-oxoethyl]-4,9,13-trioxo-3,8,11,14-tetraazaicosan-20-oate(ML-26) having the following structure is described.

Step A: benzyl{4-[(2-{[α-D-mannopyranoyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-4-oxobutyl}carbamate

To a mixture of 2-aminoethylα-D-mannopyranosyl-(1→3)]-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranoside(790 mg, 1.44 mmol) and 4-{[(benzyloxy)carbonyl]amino}butanoic acid (342mg, 1.443 mmol) in DMF (5 mL) was added EDC (553 mg, 2.89 mmol) and DMAP(176 mg). After stirring at rt overnight, the reaction mixture wasconcentrated and the residue was purified by flash chromatography on C18reverse phase silica gel (43 g), eluting 5-40% AcCN in water to give thetitle compound. UPLC Method B: m/e=767.2084 [M+1]; Rt=2.56 min.

Step B:4-amino-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)butanamide

To a nitrogen flushed solution of benzyl{4-[(2-{[α-D-mannopyranoyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-4-oxobutyl}carbamate(955 mg, 1.25 mmol) in water (6 mL) was added 10% palladium on carbon(133 mg) and the resulting mixture stirred under a balloon of H₂ for 4hr. The reaction mixture was filtered through a Celite pad, and thefiltrate was freeze-dried to give the title compound. UPLC Method B:m/e=633.2224 [M+1]; Rt=0.78 min.

Step C: 4-amino-N-{2-[(α-L-fucopyranosyl)oxy]ethyl}butanamide

The title compound was prepared using the procedure analogous to thatdescribed for ML-26 substituting 2-aminoethyl α-L-fucopyranoside for2-aminoethylα-D-mannopyranosyl-(1→3)]-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosidein Step A. UPLC Method E: m/e=1248.365 [M+1]; Rt=1.37 min.

Step D:11-[2-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-1-[(α-L-fucopyranosyl)oxy]-6-oxohexyl}amino)-2-oxoethyl]-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)-4,9,13-trioxo-3,8,11,14-tetraazaoctadecan-18-amide

The title compound was prepared using procedures analogous to thosedescribed for ML-23 substituting4-amino-N-{2-[(α-L-fucopyranosyl)oxy]ethyl}butanamide for6-amino-N-{2-[(α-L-fucopyranosyl)oxy]ethyl}hexanamide in Step C, and4-amino-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)butanamidefor 2-aminoethylα-D-mannopyranosyl-(1→3)]-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosidein Step D, respectively. UPLC Method E: m/e=1290.4012 [M+1]; Rt=2.03min.

Example 27

The synthesis of oligosaccharide linker 2,5-Dioxopyrrolidin-1-ylN-(2-{[4-({2-[(6-deoxy-α-L-galactopyranosyl)oxy]ethyl}amino)-4-oxobutyl]amino}-2-oxoethyl)-N-[2-({4-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-4-oxobutyl}amino)-2-oxoethyl]glycyl-β-alaninate(ML-27) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-23 substituting benzyl{4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutyl}carbamate for benzyl{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}carbamate in step B. UPLCMethod E: m/e=1248.365 [M+1]; Rt=1.37 min.

Example 28

The synthesis of oligosaccharide linkerN-{2-[(α-L-Fucopyranosyl)oxy]ethyl}-11-[2-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl]-1-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}-4,9,13-trioxo-3,8,11,14-tetraazaicosan-20-amide(ML-28) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-23 substituting4-amino-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)butanamidefor 2-aminoethylα-D-mannopyranosyl-(1→3)]-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosidein Step D. UPLC Method E: m/e=1318.4270 [M+1]; Rt=2.19 min.

Example 29

The synthesis of oligosaccharide linker 2,5-Dioxopyrrolidin-1-yl6-({[(2-oxo-2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]-2-oxyethyl}amino)({2-oxo-2-[(α-L-fucopyranosyl)oxy]-2-oxoethyl}amino)ethyl]amino}acetamido)-6-oxohexanoate(ML-29) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-23 substituting 2-aminoethyl α-L-fucopyranoside for6-amino-N-{2-[(α-L-fucopyranosyl)oxy]ethyl}hexanamide in Step C. UPLCMethod A: m/e=1120.30 [M+1]; Rt=1.90 min.

Example 30

The synthesis of oligosaccharide linker 2,5-Dioxopyrrolidin-1-yl2-({[(2-oxo-2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]-2-oxyethyl}amino)({2-oxo-2-[(α-L-fucopyranosyl)oxy]-2-oxoethyl}amino)ethyl]amino}acetamido)acetate(ML-30) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-23 substituting benzylN,N-bis(carboxymethyl)glycylglycinate for2,2′-((2-((6-(benzyloxy)-6-oxohexyl)amino)-2-oxoethyl)imino)diaceticacid, and 2-aminoethyl α-L-fucopyranoside for6-amino-N-(2-α-L-fucopyranosyl)ethyl)hexanamide in Step C, respectively.UPLC Method B: m/e=1064.25 [M+1]; Rt=2.65 min.

Example 31

The synthesis of oligosaccharide linker2-{[2-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl][2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-β-D-glucopyranosyl]oxy}ethyl)acetamide(ML-31) having the following structure is described.

Step A: benzyl{2-[(4,6-O-benzylidene-β-D-glucopyranosyl)oxy]ethyl}carbamate

To a solution of benzyl [2-β-D-glucopyranosyloxy)ethyl]carbamate (10 g,28.0 mmol, Beilstein J. Org. Chem. 2010, 6, 699) in AcCN (150 mL) wasadded benzaldehyde dimethyl acetal (5 mL, 31.6 mmol) andp-toluenesulfonic acid monohydrate (60 mg, 0.315 mmol). After stirringfor 24 hr, the reaction mixture was concentrated. The residue waspurified by flash chromatography on silica gel (330 g), eluting with0-20% CH₃OH in CH₂Cl₂ to give the title compound. UPLC Method B:calculated for C₂₃H₂₇NO₈ 445.17, observed m/e: 446.06 [M+1]; Rt=3.21min. ¹H NMR (CDCl₃) δ 7.50-7.45 (2H, m), 7.35-7.25 (8H, m), 5.50 (1H,s), 5.10 (2H, s), 4.40-4.36 (1H, m), 4.31-4.26 (1H, m), 3.95-3.85 (1H,m), 3.80-3.70 (2H, m), 3.55-3.40 (4 H, m), 3.40-3.30 (2H, m).

Step B: benzyl{2-[(2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl)oxy]ethyl}carbamate

A stirring mixture of benzyl{2-[(4,6-O-benzylidene-β-D-glucopyranosyl)oxy]ethyl}carbamate (4.5 g,10.10 mmol) and dibutylstannanone (3 g, 12.05 mmol) in toluene (50 mL)was allowed to reflux for 5 hr. The resulting mixture was cooled down tort and treated with benzoyl chloride (1.3 mL, 11.19 mmol). Afterstirring at rt for 1 hr, the mixture was concentrated. The residue waspurified by flash chromatography on silica gel (330 g, eluting with0-10% acetone in CH₂Cl₂) to give the title compound. UPLC Method B:calculated for C₃₀H₃₁NO₉ 549.20, observed m/e: 572.09 [M+Na]; Rt=3.94min. ¹H NMR (CDCl₃) δ 8.05-8.00 (2H, m), 7.55-7.45 (3H, m), 7.40-7.25(10H, m), 5.55 (1H, s), 5.18-5.12 (1H, m), 5.04-5.00 (1H, m), 4.93-4.89(1H, m), 4.66-4.63 (1H, m), 4.38-4.32 (1H, m), 4.05-4.00 (1H, m),3.90-3.85 (1H, m), 3.82-3.77 (1H, m), 3.70-3.60 (2H, m), 3.55-3.45 (1H,m), 3.40-3.25 (2H, m). Regiochemistry was confirmed by 1H-1H 2D COSYexperiment.

Step C: benzyl{2-[(2-O-benzoyl-4-O-benzyl-β-D-glucopyranosyl)oxy]ethyl}carbamate

To a solution of benzyl{2-[(2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl)oxy]ethyl}carbamate(2.58 g, 4.69 mmol) and borane tetrahydrofuran complex (40 mL, 40.0mmol, 1.0 M in THF) at 0° C. was added a solution ofdibutyl(((trifluoromethyl)sulfonyl)oxy)borane (6 mL, 6.00 mmol, 1.0 M inCH₂Cl₂) dropwise. After stirring at 0° C. for 2 hr, TEA (0.5 mL) wasadded to the reaction mixture and followed by the careful addition ofCH₃OH until the evolution of H₂ had ceased. The reaction mixture wasconcentrated and the residue was purified by flash chromatography onsilica gel (330 g), eluting with 0-100% EtOAc in hexanes to give thetitle compound. TLC: silica gel, hexanes/EtOAc: 35/65, R_(f)=0.5. ¹H NMR(CDCl₃) δ 8.05-8.00 (2H, m), 7.55-7.25 (13H, m), 5.05-4.98 (3H, m),4.86-4.82 (1H, m), 4.78-4.74 (1H, m), 4.60-4.58 (1H, m), 3.95-3.90 (2H,m), 3.85-3.80 (1H, m), 3.75-3.65 (2H, m), 3.61-3.58 (1H, m), 3.45-3.40(1H, m), 3.35-3.30 (2H, m). Regiochemistry was confirmed by ¹H—¹H COSYand ¹H—¹³C one-bond correlation (HSQC) 2D NMR experiments.

Step D: benzyl(2-{[2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl-(1→6)]-2-O-benzoyl-4-O-benzyl-β-D-glucopyranosyl]oxy}ethyl)carbamate

To a mixture of benzyl{2-[(2-O-benzoyl-4-O-benzyl-β-D-glucopyranosyl)oxy]ethyl}carbamate (1.47g, 2.67 mmol), 2,3,4,6-tetra-O-benzoyl-D-mannopyranosyltrichloroacetimidate (4.15 g, 5.60 mmol, Organic Letters, 2003, 5, 4041)and 4 Å molecular sieves in CH₂Cl₂ (40 mL) at −30° C. was addedtrimethylsilyl trifluoromethanesulfonate (0.25 mL, 1.384 mmol) dropwise.The mixture was allowed to gradually warm up to rt. After stirring for 6hr, the reaction was quenched with TEA (0.4 mL, 2.87 mmol). The reactionmixture was filtered and the filtrate was concentrated. The residue waspurified by flash chromatography on silica gel (330 g, eluting with0-75% EtOAc in hexanes to give the title compound. TLC: silica gel,hexanes/EtOAc 3/2, R_(f)=0.5. ¹H NMR (CDCl₃) δ 8.20-7.95 (8H, m),7.85-7.75 (8H, m), 7.65-7.60 (3H, m), 7.55-7.40 (8H, m), 7.38-7.18 (24H,m), 7.15-7.05 (4H, m), 6.00-5.95 (1H, m), 5.88-5.85 (1H, m), 5.75-5.65(3H, m), 5.48-5.46 (1H, m), 5.35-5.25 (2H, m), 5.22-5.20 (1H, m),5.07-5.05 (1H, m), 4.95-4.85 (2H, m), 4.78-4.75 (1H, m), 4.70-4.60 (1H,m), 4.60-4.55 (2H, m), 4.40-4.30 (2H, m), 4.27-4.23 (1H, m), 4.20-4.10(2H, m), 3.95-3.90 (1H, m), 3.80-3.75 (1H, m), 3.75-3.70 (3H, m),3.60-3.55 (1H, m), 3.51-3.48 (1H, m), 3.40-3.25 (2H, m).

Step E: benzyl(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-4-O-benzyl-β-D-glucopyranosyl]oxy}ethyl)carbamate

To a solution of benzyl(2-{[2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl-(1→6)]-2-O-benzoyl-4-O-benzyl-β-D-glucopyranosyl]oxy}ethyl)carbamate(3.39 g, 1.984 mmol) in CH₃OH (30 mL) was added NaOCH₃ (0.4 mL, 0.2mmol, 0.5 M in CH₃OH). After stirring at rt for 24 hr, amberlite IR 120(H) ion exchange resin (pre-washed with CH₃OH 3×30 mL) was added to thereaction mixture. The resulting mixture was allowed to stir foradditional 15 min. The resin was filtered off and washed with CH₃OH (3×5mL). The filtrate was concentrated to give the title compound. UPLCMethod B: calculated for C₃₅H₄₉NO₁₈ 771.29, observed m/e: 772.42 [M+1];Rt=2.51 min.

Step F: 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-β-D-glucopyranoside

A mixture of benzyl(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-4-O-benzyl-β-D-glucopyranosyl]oxy}ethyl)carbamate(0.96 g, 1.244 mmol) and Pd/C (124 mmol) in water (20 mL) was allowed tostir under a balloon of H₂ at rt for 16 h. The catalyst was filtered offand washed with H₂O (3×10 mL). The Titrate was concentrated to give thetitle compound. UPLC Method B: calculated for C₂₀H₃₇NO₁₆ 547.21,observed m/e: 548.29 [M+1]; Rt=0.87 min. ¹H NMR (D₂O) δ 5.20-5.19 (1H,m), 4.88-4.87 (1H, m), 4.51-4.49 (1H, m), 4.05 (1H, m), 4.00-3.90 (4H,m), 3.85-3.70 (9H, m), 3.70-3.60 (5H, m), 3.40-3.30 (1H, m), 3.05-3.00(2H, m).

Step G:2-{[2-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl][2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-β-D-glucopyranosyl]oxy}ethyl)acetamide

The title compound was prepared using procedure analogous to thosedescribed for ML-29 substituting 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-β-D-glucopyranosidefor 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranoside.UPLC Method B: calculated for C₄₄H₇₁N₅O₂₉ 1133.42, observed m/e: 1134.34[M+1]; Rt=2.17 min.

Example 32

The synthesis of oligosaccharide linker2-{[2-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl][2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-2-deoxy-2-fluoro-β-D-glucopyranosyl]oxy}ethyl)acetamide(ML-32) having the following structure is described.

Step A: 2-chloroethyl3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-D-glucopyranoside

To a solution of 2-chloroethanol (1.0 mL, 14.92 mmol),3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-D-glucopyranosyltrichloroacetimidate (1.4 g, 3.09 mmol, Angew. Chem. Int. Ed. 2010, 49,8724) and 4 Å molecular sieves in CH₂Cl₂ (50 mL) at −30° C. was addedtrimethylsilyl trifluoromethanesulfonate (0.25 mL, 1.384 mmol) dropwise.The mixture was allowed to gradually warm up to rt. After stirring for 2hr, the reaction was quenched with TEA (0.13 mL, 0.933 mmol). Theresulting mixture was filtered and the filtrate was concentrated. Theresidue was purified by flash chromatography on silica gel (80 g),eluting with 0-60% EtOAc in hexanes to give the title compound. Thisanomeric mixture was used directly in the next step without furtherpurification. TLC: silica gel, hexane/EtOAc: 3/1, R_(f)=0.35.

Step B: 2-chloroethyl 2-deoxy-2-fluoro-D-glucopyranoside

To a solution of 2-chloroethyl3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-D-glucopyranoside (0.85 g, 2.293mmol) in CH₃OH (10 mL) was added NaOCH₃ (0.46 mL, 0.230 mmol, 0.5 M inCH₃OH). The resulting mixture was stirred at rt for 2 hr. Dowex50wx2-200 (H) ion exchange resin (pre-washed with CH₃OH 3×10 mL) wasadded to the reaction mixture. After stirring for 15 min, the resin wasfiltered off and the filtrate was concentrated down to give the titlecompound. This anomeric mixture was used directly in the next stepwithout further purification. TLC: silica gel, hexane/EtOAc: 1/1,R_(f)=0.2.

Step C: 2-chloroethyl4,6-O-benzylidene-2-deoxy-2-fluoro-β-D-glucopyranoside

To a solution of 2-chloroethyl 2-deoxy-2-fluoro-D-glucopyranoside (0.55g, 2.248 mmol) in AcCN (10 mL) was added benzaldehyde dimethyl acetal(540 μL, 3.6 mmol) and p-toluenesulfonic acid monohydrate (6 mg, 0.032mmol). After stirring for 3 hr, the reaction mixture was concentrated.The residue was purified by flash chromatography on silica gel (80 g),eluting with 0 to 60% EtOAc in hexanes to give the title compound. ¹HNMR (CD₃OD) δ 7.50-7.47 (2 H, m), 7.35-7.32 (3 H, m), 5.58 (1 H, s),4.76-4.72 (1 H, m), 4.32-4.28 (1 H, m), 4.16-4.02 (2 H, m), 3.95-3.85 (2H, m), 3.80-3.74 (1 H, m), 3.70-3.66 (2 H, m), 3.52-3.48 (2 H, m). The βanomeric stereochemistry was confirmed by ¹H—¹³C one-bond correlation(HSQC) and ¹H—¹H NOE (NOESY) 2D NMR experiments. TLC: silica gel,hexane/EtOAc: 7/3, R_(f)=0.5.

Step D: 2-chloroethyl 4-O-benzyl-2-deoxy-2-floor-β-D-glucopyranoside

To a solution of 2-chloroethyl4,6-O-benzylidene-2-deoxy-2-fluoro-β-D-glucopyranoside (422 mg, 1.268mmol) in borane tetrahydrofuran complex (9 mL, 9.0 mmol, 1.0 M in THF)at 0° C. was added a solution of dibutyl{[(trifluoromethyl)sulfonyl]oxy}borane (1.27 mL, 1.270 mmol, 1.0 M inCH₂Cl₂) dropwise. After stirring at 0° C. for 2 h, TEA (0.5 mL) wasadded to the reaction mixture and followed by the careful addition ofCH₃OH until the evolution of H₂ had ceased. The reaction mixture wasconcentrated and the residue was purified by flash chromatography onsilica gel (40 g), eluting with 0-60% EtOAc in hexanes to give the titlecompound. TLC: silica gel, hexane/EtOAc: 1/1, R_(f)=0.6. ¹H NMR (CDCl₃)δ 7.38-7.28 (5 H, m), 4.84-4.71 (2H, m), 4.56-4.53 (1H, m), 4.22-4.04(2H, m), 3.93-3.83 (3 H, m), 3.77-3.71 (1H, m), 3.67-3.63 (2H, m),3.55-3.50 (1H, m), 3.40-3.37 (1 H, m). Regiochemistry was confirmed by¹H—¹³C one-bond correlation (HSQC); ¹H—¹³C multiple-bond correlation(HMQC); and ¹H—¹H NOE (NOESY) 2D NMR experiments.

Step E: 2-azidoethyl 4-O-benzyl-2-deoxy-2-fluoro-β-D-glucopyranoside

To a solution of 2-chloroethyl4-O-benzyl-2-deoxy-2-fluoro-β-D-glucopyranoside (1.53 g, 4.57 mmol) inDMF (45 mL) at rt was added sodium azide (360 mg, 5.54 mmol). Afterstirring at 70° C. for 16 hr, the reaction mixture was cooled down to rtand poured onto ice water (200 mL) and extracted with CH₂Cl₂ (3×100 mL).The organic layers were combined and washed with brine (2×100 mL), driedover Na₂SO₄, filtered and concentrated. The residue was purified byflash chromatography on silica gel (120 g), eluting with 0-100% EtOAc inhexanes to give the title compound. TLC: silica gel, hexane/EtOAc: 1/1,R_(f)=0.55. ¹H NMR (CDCl₃) δ 7.37-7.28 (5 H, m), 4.84-4.71 (2H, m),4.55-4.52 (1 H, m), 4.22-4.07 (1H, m), 4.03-3.98 (1H, m), 3.94-3.86 (2H,m), 3.79-3.71 (2H, m), 3.56-3.51 (1H, m), 3.48-3.36 (3H, m).

Step F: 2-azidoethyl2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl-(1→6)]-4-O-benzyl-2-deoxy-2-fluoro-β-D-glucopyranoside

To a solution of 2-azidoethyl4-O-benzyl-2-deoxy-2-fluoro-β-D-glucopyranoside (1.23 g, 3.6 mmol),2,3,4,6-tetra-O-benzoyl-D-mannopyranosyl trichloroacetimidate (5.35 g,7.22 mmol, Organic Letters, 2003, 5, 4041), and 4 Å molecular sieves inCH₂Cl₂ (60 mL) at -30° C. was added trimethylsilyltrifluoromethanesulfonate (0.25 mL, 1.384 mmol) added dropwise. Themixture was allowed to gradually up to rt. After stirring for 6 h, thereaction was quenched with TEA (0.4 mL, 2.87 mmol). The resultingmixture was filtered and the filtrate was concentrated. The residue waspurified by flash chromatography on silica gel (330 g), eluting with0-75% EtOAc in hexanes to give the title compound. ¹H NMR (CDCl₃) δ8.20-7.70 (16 H, m), 7.60-7.05 (29H, m), 6.14-5.98 (2H, m), 5.90-5.80(2H, m), 5.79-5.77 (1H, m), 5.66-5.64 (1H, m), 5.42-5.41 (1H, m),5.23-5.22 (1H, m), 5.03-5.02 (1H, m), 4.91-4.89 (1 H, m), 4.70-4.60 (3H,m), 4.57-4.55 (1H, m), 4.50-4.48 (1H, m), 4.40-4.22 (3H, m), 4.10-4.00(2H, m), 3.80-3.70 (3H, m), 3.55-3.45 (2H, m), 3.44-3.38 (1H, m),3.36-3.30 (1H, m).

Step G: 2-azidoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-4-O-benzyl-2-deoxy-2-fluoro-β-D-glucopyranoside

To a solution of 2-azidoethyl2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl-(1→6)]-4-O-benzyl-2-deoxy-2-fluoro-β-D-glucopyranoside(5.0 g, 3.34 mmol) in CH₃OH (40 mL) was added NaOCH₃ (1.0 mL, 0.5 mmol,0.5 M in CH₃OH). After stirring at rt for 24 hr, amberlite IR 120 (H)ion exchange resin (pre-washed with CH₃OH 3×30 mL) was added to thereaction mixture. After 15 min, the resin was filtered off and washedwith CH₃OH (3×5 mL). The filtrate was concentrated and the residue wastaking into EtOAc (50 mL) and stirred for 2 hr. The solid was filtered,and washed with EtOAc (3×15 mL) and dried to give the title compound.UPLC Method B: calculated for C₂₇H₄₀FN₃O₁₅ 665.24, observed m/e: 666.35[M+1]; Rt=2.03 min.

Step H: 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-4-O-benzyl-2-deoxy-2-fluoro-β-D-glucopyranoside

A mixture of 2-azidoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-4-O-benzyl-2-deoxy-2-fluoro-β-D-glucopyranoside(1.77 g, 2.66 mmol) and Pd/C (0.133 mmol) in water (30 mL) was allowedto stir under a balloon of H₂ at rt for 16 h. The catalyst was filteredoff and washed with H₂O (3×10 mL). The Titrate was concentrated to givethe title compound. UPLC Method B: calculated for C₂₀H₃₆FNO₁₅ 549.21,observed m/e: 550.29 [M+1]; Rt=0.86 min. ¹H NMR (D₂O) δ 5.16-5.14 (1H,m), 4.88-4.86 (1H, m), 4.80-4.76 (1H, m), 4.30-4.16 (1H, m), 4.06-4.03(1H, m), 3.98-3.88 (4H, m), 3.86-3.72 (9H, m), 3.70-3.62 (5H, m),2.94-2.88 (2H, m).

Step I:2-{[2-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl][2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-2-deoxy-2-fluoro-β-D-glucopyranosyl]oxy}ethyl)acetamide

The title compound was prepared using procedures analogous to thosedescribed for ML-29 substituting 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-4-O-benzyl-2-deoxy-2-fluoro-β-D-glucopyranosidefor 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranoside.UPLC Method B: calculated for C44H71N5O29 1133.42, observed m/e: 1134.34[M+1]; Rt=2.17 min.

Example 33

The synthesis of oligosaccharide linker2-{[2-({2-[(α-L-Fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl][2-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl]amino}-N-[2-(β-D-glucopyranosyloxy)ethyl]acetamide(ML-33) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-23 substituting 2-aminoethyl α-L-fucopyranoside for6-amino-N-(2-α-L-fucopyranosyl)ethyl)hexanamide in Step C and2-aminoethyl α-D-glucopyranoside for 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosidein Step D, respectively. UPLC Method B: m/e=796.38 [M+1]; Rt=1.87 min.

Example 34

The synthesis of oligosaccharide linker N,N-Bis{2-[(α-L-fucopyranosyl)oxy]ethyl}-6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexanamide(ML-34) having the following structure is described.

Step A: prop-2-en-1-yl 2,3,4-tri-O-benzoyl-α-L-fucopyranoside

To a stirred solution of 1,2,3,4-tetra-O-benzoyl-L-fucopyranoside (70.34g, 121 mmol, Organic Letters 2007, 9, 1227-30) in CH₂Cl₂ (300 mL) at 0°C. was added allyl alcohol (12.36 mL, 182 mmol) followed by dropwiseaddition of boron trifluoride diethyl etherate (44.9 mL, 363 mmol) over1 hr, while keeping the internal temperature below 20° C. After stirringat rt for 16 hr, the reaction mixture was chilled to 0° C., to whichsat. NaHCO₃ (600 mL, 121 mmol) was added slowly. After stirring for 16hr, the reaction mixture was extracted with CH₂Cl₂ (2×400 mL). Theorganic phase was washed with water (200 mL), sat. NaHCO₃ (3×100 mL),and brine (200 mL). The organic phase was separated, dried over MgSO₄,and filtered. The filtrate was concentrated and the residue was dividedinto five equal portions, which were separately purified by flashchromatography on silica gel (330 g), eluting with 0-60% EtOAc inhexanes to give the title compound. (α isomer R_(f)=0.63 30:70EtOAc:Hexanes). ¹H NMR (CDCl₃) δ 1.32 (3H, t, J=6.77), 2.09 (1H, s),4.15-4.14 (1H, m), 4.33-4.31 (1H, m), 4.51-4.49 (1H, m), 5.22 (1H, d,J=10.52), 5.39-5.38 (2H, m), 5.73 (1H, dd, J=10.74, 3.67), 5.82 (1H, d,J=3.30), 5.92-5.91 (1H, m), 6.04 (1H, dd, J=10.75, 3.43), 7.44 (3H, dt,J=22.64, 7.61), 7.48-7.57 (5H, m), 7.65 (1H, d, J=7.49), 7.84 (2H, d,J=7.84), 8.04 (2H, d, J=7.84), 8.15 (2H, d, J=7.79).

Step B: 2-oxoethyl 2,3,4-tri-O-benzoyl-α-L-fucopyranoside

To a solution of prop-2-en-1-yl 2,3,4-tri-O-benzoyl-α-L-fucopyranoside(6.06 g, 11.73 mmol) in acetone (94 mL) and water (23.5 mL) was added4-methylmorpholine 4-oxide (2.75 g, 23.46 mmol) followed by the additionof 2.5% OsO₄ in water (5.97 g, 0.587 mmol). The mixture was allowed tostir at rt for 16 hr. To the resulting mixture was then added a solutionof NaIO₄ (5.40 g, 23.46 mmol) in water (100 mL). After stirring foradditional 6 hr, the precipitate was filtered and washed with acetone(200 mL). The volume of the filtrate was reduced to approximately ⅓ ofthe initial volume and then extracted with EtOAc (200 mL). The organicphase was separated, washed with sat. NaHCO₃ (200 mL). The aqueous phasewas extracted with EtOAc (3×100 mL). The organic phases were combinedand washed with brine, dried over Na₂SO₄, and concentrated. The residuewas purified by flash chromatography on silica gel (220 g), eluting with0-100% EtOAc in hexanes to give the title compound. ¹H NMR (CDCl₃) δ1.33-1.29 (3H, m), 3.27 (1H, s), 3.41 (1H, s), 4.35-4.31 (1H, m),5.51-5.45 (1H, m), 5.75-5.69 (1H, m), 5.81 (1H, dd, J=13.91, 3.57),6.05-5.98 (1H, m), 7.42 (2H, d, J=7.76), 7.53 (5H, d, J=8.61), 7.67-7.63(2H, m), 7.84-7.81 (2H, m), 8.01 (2H, t, J=8.82), 8.14-8.12 (2H, m),9.77-9.77 (1H, m).

Step C: benzyl{2-[(2,3,4-tri-O-acetyl-α-L-fucopyranosyl)oxy]ethyl}carbamate

To a stirred solution of 1,2,3,4-tetra-O-acetyl-L-fucopyranose (200 g,601.86 mmol) in AcCN (100 mL) and benzyl (2-hydroxyethyl)carbamate(140.96 g, 722.08 mmol) at 0° C. was added BF₃.Et₂O (427.7 g, 3.01 mol)dropwise over 2 hr. After stirring at rt for 16 hr, the reaction mixturewas cooled to 0° C. and followed by the addition of Et₃N (130 mL). Theresulting mixture was concentrated and the residual was dissolved inCH₂Cl₂ (2.0 L), which was subsequently washed with sat. NaHCO₃ (2×500mL), water (2×500 mL) and brine (500 mL). The organic phase wasseparated, dried over Na₂SO₄, and concentrated. The residue was purifiedby flash chromatography on silica gel, eluting with EtOAc/petroleumether (1:3) to give the title compound. ¹H NMR (CDCl₃) δ 7.33-7.37(5H,m), 5.12-5.32(5H, m), 4.99-5.04(1H, m), 4.42-4.45 (1H, d), 3.87-3.94(1H, m), 3.78-3.84 (1H, m), 3.66-3.70(1H, m), 3.42-3.44 (2H, m), 2.19(3H, s), 2.05-2.12(6H, m), 1.25-1.30 (3H, d).

Step D: 2-aminoethyl 2,3,4-tri-O-acetyl-α-L-fucopyranoside

To a solution of benzyl{2-[(2,3,4-tri-O-acetyl-α-L-fucopyranosyl)oxy]ethyl}carbamate (1.0 g,2.139 mmol) in water (10 mL) was added Pd/C (68 mg, 0.642 mmol). Theresulting suspension was degassed and stirred under a balloon of H₂ atrt. After 1 hr, the reaction mixture was filtered through a Celite padand the filtrate was lyophilized to yield the title compound. UPLCMethod B: m/e=334.1563 [M+1]; Rt=1.43 min.

Step E: 2-(benzylamino)ethyl 2,3,4-tri-O-acetyl-α-L-fucopyranoside

To a solution of 2-aminoethyl 2,3,4-tri-O-acetyl-α-L-fucopyranoside(8.56 g, 25.7 mmol) in CH₂Cl₂ (100 mL) was added benzaldehyde (2.197 ml,21.67 mmol), acetic acid (372 μL, 6.50 mmol) and NaCNBH₃ (3.40 g, 54.2mmol). After stirring at rt for 16 hr, the reaction mixture wasconcentrated and the residue was partitioned between EtOAc (50 mL) andsat. NaHCO₃ (50 mL). The organic phase separated, washed with sat'dNaHCO₃ (2×100 mL), brine (100 mL), dried over MgSO₄, and concentrated.The residue was purified by flash chromatography on C18 reverse phasesilica gel (130 g), eluting with 0-100% AcCN in water to give the titlecompound. UPLC Method B: m/e=424.2089 [M+1]; Rt=3.42 min.

Step F:2-(benzyl{2-[(2,3,4-tri-O-benzoyl-α-L-fucopyranosyl)oxy]ethyl}amino)ethyl2,3,4-tri-O-acetyl-α-L-fucopyranoside

To a solution of 2-(benzylamino)ethyl2,3,4-tri-O-acetyl-α-L-fucopyranoside (1.021 g, 2.411 mmol) in CH₂Cl₂(50 mL) was added 2-oxoethyl 2,3,4-tri-O-benzoyl-α-L-fucopyranoside(1.25 g, 2.411 mmol), acetic acid (41 μL, 0.723 mmol) and NaCNBH₃ (227mg, 3.62 mmol). After stirring at rt for 16 hr, the reaction mixture wasconcentrated and the residue was partitioned between EtOAc (50 mL) andsat'd NaHCO₃ (50 mL). The organic phase was separated, washed withsaturated NaHCO₃ (2×100 mL) and brine (100 mL), dried over MgSO₄, andconcentrated. The residue was purified by flash chromatography silicagel (120 g, eluting 0-100 EtOAc in hexanes). The fractions containingthe title compound were combined and concentrated. The residue wasfurther purified by flash chromatography on C18 reverse phase silica gel(130 g), eluting with 0-100% AcCN in water to give the title compound.UPLC Method B: me/e=926.3234 [M+1]; Rt=1.947 min. ¹H NMR (CDCl₃) δ 1.12(3H, d, J=6.54), 1.27 (3H, d, J=6.57), 2.00 (6H, d, J=5.40), 2.20 (3H,s), 2.78 (2H, q, J=5.85), 2.85 (2H, t, J=5.72), 3.48-3.46 (1H, m),3.77-3.59 (4H, m), 3.87-3.85 (1H, m), 4.09 (1H, d, J=6.63), 4.39 (1H, d,J=6.71), 5.02 (1H, d, J=3.71), 5.14 (1H, dd, J=10.82, 3.68), 5.30 (1H,d, J=3.36), 5.37-5.34 (2H, m), 5.65 (1H, dd, J=10.72, 3.66), 5.77 (1H,d, J=3.49), 5.98 (1H, dd, J=10.70, 3.47), 7.29 (6H, s), 7.36 (3H, t,J=7.74), 7.45 (1H, t, J=7.59), 7.52 (3H, t, J=7.73), 7.64 (1H, t,J=7.46), 7.81 (2H, dd, J=8.00, 1.41), 7.97 (2H, dd, J=8.07, 1.40),8.14-8.12 (2H, m).

Step G: 2-(benzyl{2-[(α-L-fucopyranosyl)oxy]ethyl}amino)ethyl6-α-L-fucopyranoside

To a solution of 2-(benzyl{2-[(2,3,4-tri-O-benzoyl-α-L-fucopyranosyl)oxy]ethyl}amino)ethyl2,3,4-tri-O-acetyl-α-L-fucopyranoside (432.9 mg, 0.468 mmol) in CH₃OH(10 mL) was added NaOCH₃ (0.087 μL, 0.468 mmol, 1.0 M). After stirringfor 16 hr, the reaction mixture was concentrated to give the titlecompound. UPLC Method B: m/e=488.2324 [M+1]; Rt=2.106 min.

Step H: 2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)ethylα-L-fucopyranoside

To a solution of 2-(benzyl{2-[(α-L-fucopyranosyl)oxy]ethyl}amino)ethyl6-α-L-fucopyranoside (220 mg, 0.451 mmol) in water (10 mL) was addedPd/C (14.41 mg, 0.135 mmol). The mixture was degassed and stirred undera balloon of H₂. After 1 hr, the reaction mixture was filtered through aCelite pad and the filtrate was lyophilized to yield the title compound.UPLC Method: m/e=398.2161 [M+1]; Rt=1.119 min. ¹H NMR (CD₃OD) δ 1.24(6H, d, J=6.58), 2.91-2.89 (4H, m), 3.56 (2H, ddd, J=10.66, 6.64, 4.66),3.70-3.69 (2H, m), 3.80-3.75 (4H, m), 3.86 (2H, dt, J=10.61, 4.53), 3.98(2H, q, J=6.63), 4.80 (2H, d, J=3.45).

Step I: benzyl6-(bis{2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoate

To a solution of 6-(benzyloxy)-6-oxohexanoic acid (40 mg, 0.169 mmol),EDC (114 mg, 0.593 mmol) and HOBt (2.59 mg, 0.017 mmol) in DMF (5 mL) atrt was added 2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)ethylα-L-fucopyranoside (190 mg, 0.478 mmol). After stirring for 16 hr, thereaction mixture was concentrated and the residue was purified by flashchromatography on C18 reverse phase silica gel (50 g), eluting with5-40% AcCN in water to give the title compound. UPLC Method B:m/e=616.2923 [M+1]; Rt=3.114 min. ¹H NMR (CD₃OD) δ 1.24 (6H, d, J=6.58),1.72-1.64 (4H, m), 2.45 (2H, t, J=7.11), 2.53 (2H, t, J=7.32), 3.91-3.55(17H, m), 4.78-4.75 (2H, m), 5.14 (2H, s), 7.38-7.37 (5H, m).

Step J:N,N-Bis{2-[(α-L-fucopyranosyl)oxy]ethyl}-6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexanamide

The title compound was prepared using procedure analogous to thosedescribed for ML-1 substituting benzyl 6-(bis{2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-6-oxohexanoate for benzyl6-({2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}amino)-6-oxohexanoatein Step C. UPLC Method B: m/e=623.2853 [M+1]; Rt=2.155 min.

Example 35

The synthesis of oligosaccharide linker2-({2-[(α-L-Fucopyranosyl)oxy]ethyl}{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)ethylα-L-fucopyranoside (ML-35) having the following structure is described.

Step A: benzyl6-(bis{2-[(2,3,4-tri-O-benzoyl-α-L-fucopyranosyl)oxy]ethyl}amino)hexanoate

To a solution of 2-oxoethyl 2,3,4-tri-O-benzoyl-α-L-fucopyranoside (1.25g, 2.411 mmol) in CH₂Cl₂ (50 mL) was added6-(benzyloxy)-6-oxohexan-1-aminium 4-methylbenzenesulfonate, acetic acid(17 μL, 0.300 mmol) and NaCNBH₃ (189 mg, 3.00 mmol). After stirring atrt for 16 hr, the reaction mixture was concentrated and the residue waspartitioned between EtOAc (50 mL) and sat'd NaHCO₃ (50 mL). The organicphase was separated, washed with sat'd NaHCO₃ (2×100 mL), brine (100mL), dried over MgSO₄, and concentrated. The residue was purified byflash chromatography on silica gel (120 g), eluting with 0-100% EtOAc inhexanes. The fractions containing the title compound were combined andconcentrated. The residue was further purified by flash chromatographyon C18 reverse phase silica gel (50 g), eluting with 0-100% AcCN inwater to give the title compound. UPLC Method B: m/e=1226.4591 [M+1];Rt=3.310 min. ¹H NMR (CDCl₃) δ 1.29 (5H, d, J=6.62), 2.36-2.31 (2H, m),2.75 (3H, d, J=23.31), 3.53 (2H, d, J=9.26), 3.78-3.76 (1H, m), 4.45(1H, d, J=6.66), 5.13 (2H, s), 5.35 (1H, d, J=3.64), 5.64 (2H, dd,J=10.69, 3.63), 5.79 (2H, d, J=3.48), 5.98 (1H, dd, J=10.71, 3.44), 7.26(4H, t, J=7.64), 7.56-7.40 (14H, m), 7.64 (2H, t, J=7.72), 7.82-7.80(5H, m), 7.99-7.97 (5H, m), 8.19-8.12 (5H, m).

Step B: methyl 6-(bis{2-[(α-L-fucopyranosyl)oxy]ethyl}amino)hexanoate

The title compound was prepared using procedures analogous to thosedescribed for ML-34 in step G substituting benzyl 6-(bis{2-[(2,3,4-tri-O-benzoyl-α-L-fucopyranosyl)oxy]ethyl}amino)hexanoate for2-(benzyl{2-[(2,3,4-tri-O-benzoyl-α-L-fucopyranosyl)oxy]ethyl}amino)ethyl2,3,4-tri-O-acetyl-α-L-fucopyranoside. UPLC Method B: m/e=526.2852[M+1]; Rt=2.112 min.

Step C: 6-(bis{2-[(α-L-fucopyranosyl)oxy]ethyl}amino)hexanoic acid

To a solution of methyl6-(bis{2-[(α-L-fucopyranosyl)oxy]ethyl}amino)hexanoate (45 mg, 0.086mmol) in water (10 mL) was added NaOH (0.086μL, 0.086 mmol, 1.0 M).After stirring for 16 hours, the reaction mixture was neutralized with0.01 M HCl and the resulting solution was lyophilized to yield the titlecompound. UPLC Method B: m/e=512.2866 [M+1]; Rt=1.709 min.

Step D:2-({2-[(α-L-fucopyranosyl)oxy]ethyl}{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)ethylα-L-fucopyranoside

The title compound was prepared using procedures analogous to thosedescribed for ML-1 Step D, substituting6-(bis{2-[(α-L-fucopyranosyl)oxy]ethyl}amino)hexanoic acid for6-({2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}amino)-6-oxohexanoicacid. UPLC Method B: m/e=609.2808 [M+1]; Rt=2.088 min.

Example 36

The synthesis of oligosaccharide2-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}[3-(α-L-fucopyranosyl)propyl]amino)ethyl2-(acetylamino)-2-deoxy-β-D-glucopyranoside (ML-36) having the followingstructure is described.

Step A: 3-(2,3,4-tri-O-acetyl-α-L-fucopyranosyl)-1-propene

To a solution of 1,2,3,4-tetra-O-acetyl-α-L-fucopyranose (12 g, 36.1mmol) and allyltrimethyl silane (11.48 mL, 72.2 mmol) in AcCN (60 mL) at0° C. was added TMS-OTf (3.52 mL, 19.50 mmol). The reaction mixture wasstirred at 0° C. for 18 hr and then at rt for 6 hr. The resulting redsolution was diluted with CH₂Cl₂ (250 mL) and sat'd NaHCO₃ (150 mL) wasadded carefully. The aqueous layer was separated and extracted withCH₂Cl₂ (2×50 mL). The combined organic layers were dried over Na₂SO₄,filtered and concentrated. The residue was purified by flashchromatography on silica gel (220 g), eluting with 15% EtOAc in hexanesto give the title compound. ¹H NMR (CDCl₃, 400 MHz) δ 1.16 (d, J=6.4,3H), 2.04 (s, 3H), 2.08 (s, 3H), 2.18 (s, 3H), 2.34 (m, 1H), 2.57 (m,1H), 4.00 (m, 1H), 4.29 (dt, J=10.4, 7.3, 1H), 5.13 (m, 2H), 5.23 (dd,J=10.0, 3.4, 1H), 5.30 (dd, J=3.4, 1.9, 1H), 5.35 (dd, J=10.0, 5.6, 1H),5.77 (m, 1H).

Step B: 3-(α-L-fucopyranosyl)-1-propene

To a stirred solution of3-(2,3,4-tri-O-acetyl-α-L-fucopyranosyl)-1-propene (10.65 g, 33.9 mmol)in CH₃OH (50 mL) was added NaOCH₃ (183 mg, 3.4 mmol). After stirring atrt for 2 hr, the reaction mixture was neutralized with Amberlite IR120(pre-washed with methanol 3×25 mL). The resin was filtered off and thefiltrate was concentrated to give a white solid, which wasrecrystallized from EtOAc (˜200 mL) to give the title compound. ¹H NMR(CDCl₃, 400 MHz) δ 1.22 (d, J=6.5, 3H), 2.41 (m, 1H), 2.47 (m, 1H), 3.73(m, 2H), 3.85 (qd, J=6.5, 2.0, 1H), 3.90 (dd, J=8.9, 5.5, 1H), 3.99 (m,1H), 5.07 (m, 1H), 5.15 (dq, J=17.2, 1.7, 1H), 5.85 (m, 1H).

Step C: 3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-1-propene

To a stirred suspension of NaH (3.91 g, 60% dispersion in oil, 98 mmol)in DMF (120 mL) at rt was added portionwise3-(α-L-fucopyranosyl)-1-propene (4.6 g, 24.44 mmol). After 2 hr, to theresulting mixture was added tetrabutyl ammonium iodide (451 mg, 1.22mmol) and followed by the slow addition of benzyl bromide (13.1 mL, 110mml) After stirring at rt for 16 hr, the reaction mixture wasconcentrated and the residue was partitioned between water (300 mL) andEt₂O (150 mL). The aqueous layer was extracted with Et₂O (3×150 mL). Thecombined organic layers were washed with brine (100 mL), dried overNa₂SO₄, filtered and concentrated. The residue was purified by flashchromatography on silica gel (220 g), eluting with 0-40% EtOAc inhexanes to give the title compound. ¹H NMR (CDCl₃, 400 MHz) δ 1.34 (d,J=6.6, 3H), 2.33 (m, 1H), 2.42 (m, 1H), 3.82 (m, 3H), 3.99 (m, 1H), 4.12(m, 1H), 4.58 (d, J=11.8, 1H), 4.64 (d, J=11.8, 2H), 4.69 (d, J=12.0,1H), 4.76 (dd, J=12.0, 8.9, 2H), 5.05, 5.07 and 5.11 (m, 2H), 5.80 (m,1H), 7.30-7.40 (m, 15H).

Step D: 3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)propanol

A solution of 3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-1-propene (10.4g, 22.68 mmol) in THF (100 mL) at 0° C. was added slowly 9-BBN (58.5 mL,29.3 mmol, 0.5 M in THF). The mixture was allowed to warm up to rt, andthen refluxed for 3 hr. The reaction mixture was then cooled to rt andethanol (4.4 mL, 75 mmol) was added dropwise, followed by NaOH (11.51ml, 46 mmol, 4.0 M in water). The resulting mixture was cooled to 0° C.and 35% hydrogen peroxide (10 mL, 115 mmol) was added. The resultingsuspension was stirred at rt overnight. The reaction mixture was dilutedwith brine (125 mL) and ether (200 mL). The organic layer was washedwith brine (2×125 mL), dried over Na₂SO₄, filtered and concentrated. Theresidue was purified by flash chromatography on silica gel (330 g),eluting with 0-100% EtOAc in hexanes to give the title compound. ¹H NMR(CDCl₃, 400 MHz) δ 1.33 (d, J=6.6, 3H), 1.67 (m, 3H), 1.70 (m, 1H), 3.65(m, 2H), 3.80 (m, 3H), 3.98 (m, 1H), 4.02 (m, 1H), 4.56 (d, J=11.8, 1H),4.63 (t, J=12.2, 2H), 4.69 (d, J=12.0, 1H), 4.78 (dd, J=12.1, 2.1, 2H),7.27-7.40 (m, 15H).

Step E: 3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)propanal

To solution of 3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)propanol (8.4 g,17.62 mmol) in CH₂Cl₂ (100 mL) at 0° C. was added Dess-Martinperiodinane (11.21 g, 26.4 mmol). The resulting mixture was allowed tostir at 0° C. for 1 hr and then at rt for 2 hr. TLC indicates still somestarting alcohol present so further Dess-Martin periodinane (5 g, 11.8mmol) added and the mixture was stirred at rt for additional 2 hr. Theresulting mixture was washed with sat'd NaHCO₃ (3×150 mL), brine (50mL), dried over Na₂SO₄, filtered and concentrated. The residue purifiedby flash chromatography on silica gel (220 g), eluting with 0-80% EtOAcin hexanes to give the title compound. ¹H NMR (CDCl₃, 400 MHz) δ 1.26(d, J=6.6, 3H), 1.82 (m, 1H), 2.06 (m, 1H), 2.40-2.58 (m, 2H), 3.80 (m,2H), 3.84 (m, 1H), 3.90 (m, 1H), 3.99 (dt, J=10.9, 3.8, 1H), 4.55 (d,J=11.8, 1H), 4.65 (d, J=11.8, 1H), 4.70 (t, J=12.0, 2H), 4.79 (dd,J=12.0, 9.2, 2H), 7.28-7.40 (m, 15H).

Step F: 2-{[3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)propyl]amino}ethyl2-(acetylamino)-2-deoxy-β-D-glucopyranoside

To a mixture of 3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)propanal (800mg, 1.69 mmol) and 2-aminoethyl3,4,6-tri-O-acetyl-2-(acetylamino)-2-deoxy-β-D-glucopyranoside (987 mg,2.53 mmol) in CH₂Cl₂ (15 mL) was added acetic acid (29 μL, 0.506 mmol)and sodium triacetoxyborohydride (893 mg, 4.21 mmol). After stirring atrt overnight, the reaction mixture was concentrated and the residue wastaken up in EtOAc (70 mL), washed with sat'd NaHCO₃ (2×100 mL), brine(30 mL), dried over Na₂SO₄, filtered and concentrated. The residue wastaken up in CH₃OH (8 mL), to which NaOCH₃ (27 mg, 0.506 mmol) was added.After stirring at rt for 2 hr, the resulting mixture was concentratedand the residue was purified by flash chromatography on C18 reversephase silica gel (120 g), eluting with 5-100% AcCN in water to give thetitle compound. ¹H NMR (CDCl₃, 400 MHz) δ 1.30 (d, J=6.6, 3H), 1.50 (m,1H), 1.60 (m, 1H), 1.68 (m, 1H), 2.02 (s, 3H), 2.64 (m, 2H), 2.81 (m,2H), 3.39 (m, 1H), 3.57 (m, 2H), 3.69 (m, 1H), 3.78-3.85 (m, 4H), 3.92(m, 2H), 4.00 (m, 2H), 4.45 (d, J=7.7, 1H), 4.52 (d, J=11.9, 1H), 4.62(d, J=11.8, 1H), 4.68 (d, J=12.1, 1H), 4.79 (d, J=12.0, 2H), 7.28-7.38(m, 15H), 7.65 (s, 1H); [M+H/e]+=723.3925.

Step G: benzyl6-{[3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)propyl](2-{[2-(acetylamino)-2-deoxy-β-D-glucopyranosyl]oxy}ethyl)amino}hexanoate

To a mixture of benzyl 6-oxohexanoate (170 mg, 0.77 mmol) and2-{[3-(2,3,4-tri-O-benzyloxy-α-L-fucopyranosyl)propyl]amino}ethyl2-(acetylamino)-2-deoxy-β-D-glucopyranosid (558 mg, 0.77 mmol) in CH₂Cl₂(8 mL) was added acetic acid (13 μL, 0.232 mmol) and sodiumtriacetoxyborohydride (327 mg, 1.54 mmol) and the resulting mixturestirred at room temperature for 2 hours. Further benzyl 6-oxohexanoate(170 mg, 0.77 mmol) added and stirring continued overnight. The mixtureevaporated and the residue was partitioned between EtOAc (40 mL) andsat'd NaHCO₃ (60 mL); the organic layer was washed with brine (30 mL),dried over Na₂SO₄, filtered and concentrated. The residue was purifiedby flash chromatography on silica gel (40 g), eluting with 5-20% MeOH inCH₂Cl₂ to give the title compound. ¹H NMR (CDCl₃, 400 MHz): 1.27-1.38(m, 5H), 1.48 (m, 1H), 1.62-1.75 (m, 4H), 1.80 (m, 1H), 2.09 (s, 3H);2.37 (t, J=7.3, 2H), 2.90-3.02 (m, 4H), 3.04 (m, 1H), 3.13 (m, 1H), 3.42(m, 1H), 3.59 (t, J=8.9, 1H), 3.65-3.75 (m, 3H), 3.78 (d, J=4.9, 2H),3.86 (m, 1H), 3.87-4.05 (m, 4H), 4.15 (d, J=11.2, 1H), 4.49 (d, J=11.9,1H), 4.62 (d, J=11.8, 1H), 4.66 (d, J=11.7, 1H), 4.69 (m, 2H), 4.74 (d,J=11.8, 1H), 4.78 (d, J=12.0, 1H), 5.12 (s, 2H), 7.25-7.38 (m, 15H),8.36 (s, 1H); UPLC-MS [M+H/e]+=927.5049.

Step H:2-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}[3-(α-L-fucopyranosyl)propyl]amino)ethyl2-(acetylamino)-2-deoxy-β-D-glucopyranoside

The title compound was prepared using procedures analogous to thosedescribed for Example 1, ML-1, substituting benzyl6-{[3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)propyl](2-{[2-(acetylamino)-2-deoxy-β-D-glucopyranosyl]oxy}ethyl)amino}hexanoatefor benzyl6-({2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}amino-6-oxohexanoate”in Step C. UPLC Method B: m/e=664.3474 [M+1]; Rt=1.08 min

Example 37

The synthesis of oligosaccharide linker2-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}[3-(α-L-fucopyranosyl)propyl]amino)ethylβ-D-glucopyranoside (ML-37) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-36 substituting 2-aminoethyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside for 2-aminoethyl3,4,6-tri-O-acetyl-2-(acetylamino)-2-deoxy-β-D-glucopyranoside in StepF. UPLC Method B: m/e=623.3277 [M+1]; Rt=1.11 min.

Example 38

The synthesis of oligosaccharide linker2-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}[3-(α-L-fucopyranosyl)propyl]amino)ethylα-D-glucopyranoside (ML-38) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-36 substituting 2-aminoethyl2,3,4,6-tetra-O-acetyl-α-D-glucopyranoside for 2-aminoethyl3,4,6-tri-O-acetyl-2-(acetylamino)-2-deoxy-β-D-glucopyranoside Step F.UPLC Method B: m/e=623.3336 [M+1]; Rt=1.11 min.

Example 39

The synthesis of oligosaccharide linker 2,5-Dioxopyrrolidin-1-yl6-{[3-(α-L-fucopyranosyl)propyl][2-(α-D-glucopyranosyl)propyl]amino}hexanoate(ML-39) having the following structure is described.

Step A: methyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranoside

To a suspension of NaH (5.19 g of a 60% dispersion in oil, 130 mmol) inDMF (150 mL) was added portionwise methyl-α-D-glucopyranoside (4.2 g,21.6 mmol). The resulting mixture was stirred at rt for 2 hr, to whichtetrabutyl ammonium bromide (800 mg, 2.16 mmol) was and followed bydropwise addition of benzyl bromide (11.58 mL, 97 mmol). After stirringat rt overnight, the mixture was concentrated and the residue wassuspended in water and extracted with ether (3×150 mL). The combinedether layers were washed with brine (200 mL), dried over Na₂SO₄,filtered and evaporated. The residue was purified by flashchromatography on silica gel (330 g), eluting with 0-30% EtOAc inhexanes to give the title compound. ¹H NMR (CDCl₃, 400 MHz) δ 3.44 (s,3H), 3.62 (dd, J=9.6, 3.5, 1H), 3.66-3.72 (m, 2H), 3.75-3.82 (m, 2H),4.04 (t, J=9.3, 1H), 4.51-4.55 (m, 2H), 4.66 (d, J=12.1, 1H), 4.69 (d,J=3.5, 1H), 4.72 (d, J=12.1, 1H), 4.83-4.90 (m, 3H), 5.04 (d, J=11.0,1H), 7.19 (m, 2H), 7.30-7.43 (m, 18H).

Step B: 3-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-1-propene

The title compound was prepared using the procedure analogous to thatdescribed for ML-36 in Step A, substituting methyl2,3,4,6-tetra-O-benzyl-α-D-glucopyranoside for1,2,3,4-tetra-O-acetyl-α-L-fucopyranose. ¹H NMR (CDCl₃) δ 2.48-2.60 (m,2H), 3.64-3.70 (m, 3H), 3.76 (dd, J=10.5, 3.2, 1H), 3.80-3.88 (m, 2H),4.18 (m, 1H), 4.52 (d, J=10.5, 2H), 4.68 (d, J=13.7, 2H), 4.74 (d,J=11.6, 1H), 4.86 (dd, J=10.6, 3.3, 2H), 4.98 (d, J=11.0, 1H), 5.11-5.18(m, 2H), 5.84-5.90 (m, 1H), 7.18 (m, 2H), 7.29-7.41 (m, 18H).

Step C: 3-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)propanol

The title compound was prepared using the procedure analogous to thatdescribed for ML-36 in Step D, substituting3-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-1-propene for3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-1-propene. ¹H NMR (CDCl₃) δ1.70 (m, 2H), 1.85 (m, 2H), 3.61 (m, 1H), 3.65-3.76 (m, 2H), 3.76-3.86(m, 2H), 4.91 (m, 1H), 4.52 (d, J=10.8, 1H), 4.55 (d, J=12.3, 1H), 4.65(d, J=12.1, 1H), 4.67 (d, J=11.8, 1H), 4.75 (d, J=11.7, 1H), 4.86 (d,J=11.3, 2H), 4.98 (d, J=11.0, 1H), 7.18 (m, 2H), 7.29-7.40 (m, 18H).

Step D: 3-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)propylmethanesulfonate

To a solution of 3-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)propanol(3.35 g, 5.75 mmol) in CH₂Cl₂ (30 mL) at 0° C. was added DIPEA (1.25 mL,7.19 mmol) and followed by dropwise addition of methanesulfonyl chloride(538μL, 6.9 mmol). After stirring at 0° C. for 1 hr, the reactionmixture was poured into water (50 mL). The organic layer was separatedand washed with sat'd NaHCO₃ (50 mL), brine (30 mL), dried over MgSO₄,filtered and concentrated to give the title compound. ¹H NMR (CDCl₃) δ1.76-1.90 (m, 3H), 1.90-1.99 (m, 2H), 3.00 (s, 3H), 3.58-3.66 (m, 2H),3.68-3.74 (m, 2H), 3.77-3.85 (m, 2H), 4.06 (m, 1H), 4.52 (d, J=10.7,1H), 4.54 (d, J=12.1, 1H), 4.65 (d, J=12.1, 1H), 4.66 (d, J=11.6, 1H),4.76 (d, J=11.6, 1H), 4.85 (d, J=10.9, 1H), 4.98 (d, J=10.9, 1H), 7.18(m, 2H), 7.30-7.40 (m, 18H).

Step E: 3-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)propyl azide

To a solution of 3-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)propylmethanesulfonate (3.78 g, 5.72 mmol) in AcCN (50 mL) was addedtetrabutylammonium azide (1.66 g, 5.83 mmol). The resulting mixtureallowed to reflux overnight. After cooled to rt, the reaction mixturewas concentrated. The residue was dissolved in ether (50 mL), which waswashed with water (2×50 mL), brine (25 mL), dried over Na₂SO₄, filteredand concentrated. The residue was purified by flash chromatography onsilica gel (120 g, eluting with 0-30% EtOAc in hexanes) to give thetitle compound. ¹H NMR (CDCl₃) δ 1.58-1.68 (m, 2H), 1.74-1.86 (m, 2H),3.32-3.41 (m, 2H), 3.58-3.76 (m, 4H), 3.76-3.85 (m, 2H), 4.40 (m, 1H),4.52 (t, J=10.4, 2H), 4.65 (dd, J=11.7, 2.5, 2H), 4.75 (d, J=11.7, 1H),4.86 (m, 2H), 4.97 (d, J=10.8, 1H), 7.16 (m, 2H), 7.28-7.40 (m, 18H).

Step F: 3-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)propylamine

To a nitrogen flushed solution of3-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)propyl azide (3 g, 4.94mmol) in CH₃OH (100 mL) was added 10% Pd/C (525 mg). The resultingmixture was allowed to stir under a balloon of H₂ overnight. Thereaction mixture was filtered through a Celite pad and the filtrate wasconcentrated to give the title compound. ¹H NMR (CDCl₃) δ 1.48 (m, 1H),1.59 (m, 1H), 2.23 (m, 1H), 2.30 (m, 2H), 3.66-3.76 (m, 5H), 4.00 (m,1H), 4.09 (m, 1H), 4.38 (d, J=10.0, 1H), 4.50 (d, J=12.1, 1H), 4.61 (d,J=11.7, 1H), 4.68 (d, J=11.8, 1H), 4.70 (d, J=12.0, 1H), 4.79 (d,J=10.0, 1H), 4.86 (d, J=11.2, 1H), 5.02 (d, J=11.2, 1H), 7.00 (m, 2H),7.25-7.40 (m, 18H), 8.06 (s, 2H).

Step G: 2,5-dioxopyrrolidin-1-yl6-{[3-(α-L-fucopyranosyl)propyl][2-(α-D-glucopyranosyl)propyl]amino}hexanoate

The title compound was prepared using procedures analogous to thosedescribed for ML-36 substituting3-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)propylamine for2-aminoethyl3,4,6-tri-O-acetyl-2-(acetylamino)-2-deoxy-β-D-glucopyranoside in StepF. UPLC Method B: m/e=621.3424 [M+1]; Rt=1.08 min.

Example 40

The synthesis of oligosaccharide linker 2,5-Dioxopyrrolidin-1-yl6-{[3-(α-L-fucopyranosyl)propyl][2-(β-D-glucopyranosyl)propyl]amino}hexanoate(ML-40) having the following structure is described.

Step A: 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide

To β-D-glucose pentaacetate (5 g, 12.81 mmol) was added 33% HBr inacetic acid (30 mL, 192 mmol) at rt. After stirring for 40 min, themixture was diluted with CH₂Cl₂ (150 mL) and washed with ice cold wateruntil the washings were neutral pH. The organic layer was dried overMgSO₄, filtered and concentrated to give the title compound. ¹H NMR(CDCl₃) δ 2.06 (s, 3H), 2.08 (s, 3H), 2.12 (s, 3H), 2.13 (s, 3H), 4.15(d, J=11.1, 1H), 4.31-4.37 (m, 2H), 4.86 (dd, J=9.9, 4.0, 1H), 5.19 (t,J=9.8, 1H), 5.58 (t, J=9.8, 1H), 6.63 (d, J=4.0, 1H).

Step B: 3-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-1-propene

To a solution of allyl magnesium bromide (100 mL, 100 mmol, 1.0 M inether,) at 0° C. was added dropwise2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (4.45 g, 10.82 mmol)in ether (60 mL) over a period of 1 hr. After completion of theaddition, the mixture was allowed to warm up and stirred at rtovernight. To the resulting mixture was carefully added water (200 mL)and followed by the addition of acetic acid to dissolve magnesium salts.Th organic layer was separated and concentrated. The residue was treatedwith acetic anhydride (70 mL, 740 mmol) and pyridine (100 mL). Afterstirring at rt overnight, the mixture was concentrated and the residuetaken up in EtOAc (200 mL) and washed with sat'd NaHCO₃ (5×300 mL),dried over MgSO₄, filtered and evaporated. The residue was purified byflash chromatography on silica gel (120 g, eluting with 0-100% EtOAc inhexanes) to give the title compound. ¹H NMR (CDCl₃) δ 2.00 (s, 3H), 2.03(s, 3H), 2.04 (s, 3H), 2.09 (s, 3H), 2.26-2.37 (m, 2H), 3.51 (m, 1H),3.65 (m, 1H), 4.10 (dd, J=12.2, 2.2, 1H), 4.24 (dd, J=12.2, 5.0, 1H),4.93 (t, J=9.4, 1H), 5.07 (m, 2H), 5.09 (s, 1H), 5.17 (t, J=9.4, 1H),5.83 (m, 1H).

Step C: 3-(β-D-glucopyranosyl)-1-propene

To a solution of 3-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-1-propene(4.15 g, 11.14 mmol) in CH₃OH (50 mL) was added NaOCH₃ (0.56 mL, 2.2mmol, 4.0 M in CH₃OH). After stirring at rt for 2 hr, the reactionmixture was neutralized using Dowex 50W (acidic form). The resin wasfiltered off and the filtrate was concentrated to give the titlecompound. ¹H NMR (DMSO-d6) δ 2.09 (m, 1H), 2.49 (m, 1H), 2.88 (t, J=8.9,1H), 2.98-3.07 (m, 3H), 3.11 (m, 1H), 3.39 (dd, J=11.4, 4.7, 1H), 3.60(d, J=11.6, 1H), 4.99 (dd, J=10.3, 0.9, 1H), 5.06 (d, J=17.2, 1H), 5.90(m, 1H).

Step D: 3-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)-1-propene

The title compound was prepared using the procedure analogous to thatdescribed for ML-36 in Step C, substituting3-(β-D-glucopyranosyl)-1-propene for 3-(α-L-fucopyranosyl)-1-propene. ¹HNMR (CDCl₃) δ 2.39 (m, 1H), 2.65 (m, 1H), 3.41 (m, 2H), 3.49 (m, 1H),3.68 (t, J=9.5, 1H), 3.72-3.82 (m, 3H), 4.72 (dd, J=10.8, 2.1, 1H), 4.89(d, J=10.7, 1H), 4.94-500 (m, 3H), 5.16 (m, 2H), 6.03 (m, 1H), 7.25 (m,2H), 7.32-7.44 (m, 18H).

Step E: 3-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)-1-propanol

The title compound was prepared using the procedure analogous to thatdescribed for ML-36 in Step D, substituting3-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)-1-propene for3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-1-propene. ¹H NMR (CDCl₃) δ1.60 (m, 1H), 1.76 (m, 2H), 2.04 (m, 1H), 2.36 (t, J=5.7, 1H), 3.35 (m,2H), 3.49 (m, 1H), 3.62-3.72 (m, 4H), 3.72-3.77 (m, 2H), 4.58 (d,J=12.2, 1H), 4.60 (d, J=10.7, 1H), 4.65 (d, J=12.2, 1H), 4.70 (d,J=10.8, 1H), 4.86 (d, J=10.8, 1H), 4.95 (m, 3H), 7.21 (m, 2H), 7.31-7.41(m, 18H).

Step F: 2,5-Dioxopyrrolidin-1-yl6-{[3-(α-L-fucopyranosyl)propyl][2-(β-D-glucopyranosyl)propyl]amino}hexanoate

The title compound was prepared using procedures analogous to thosedescribed for ML-39 substituting3-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)-1-propanol for3-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)propanol in Step D. UPLCMethod B: m/e=621.3412 [M+1]; Rt=1.08 min.

Example 41

The synthesis of oligosaccharide linker2-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}[3-(α-D-mannopyranosyl)propyl]amino)ethylα-L-fucopyranoside (ML-41) having the following structure is described.

Step A: 3-(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl)-1-propene

The title compound was prepared using the procedure analogous to thatdescribed for ML-39 in Step B, substituting methyl2,3,4,6-tetra-O-benzyl-α-D-mannopyranoside for methyl2,3,4,6-tetra-O-benzyl-α-D-glucopyranoside, as a mixture of α and βanomers. These anomers were separated by Chiral SFC chromatography(Column: AD-H (50×250 cm), eluting with 30% IPA (0.1% DEA)/CO₂, 100 bar,200 mL/min, 220 nm; injection volume: 1.0 mL at concentration of 162mg/mL 4:1 v/v IPA/CH₂Cl₂) to give the major product as the α anomer. ¹HNMR (CDCl₃) δ 2.38 (m, 2H), 3.68 (dd, J=4.6, 3.2, 1H), 3.76 (dd, J=10.4,3.7, 1H), 3.83 (m, 2H), 3.90 (m, 2H), 4.10 (m, 1H), 4.55-4.62 (m, 4H),4.62-4.65 (m, 3H), 4.75 (d, J=11.3, 1H), 5.06 (d, J=4.6, 1H), 5.08 (s,1H), 5.80 (m, 1H), 7.25 (m, 2H), 7.30-7.42 (m, 18H).

Step B: 3-(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl)-1-propanol

The title compound was prepared using the procedure analogous to thatdescribed for ML-39 in Step C, substituting3-(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl)-1-propene for3-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-1-propene. ¹H NMR (CDCl₃)δ 1.85-2.00 (m, 2H), 2.53 (m, 1H), 2.60 (m, 1H), 3.62 (dd, J=6.0, 2.4,1H), 3.73 (dd, J=10.2, 4.2, 1H), 3.78-3.87 (m, 3H), 3.90 (m, 1H), 3.98(m, 1H), 4.54-4.62 (m, 7H), 4.67 (d, J=11.5, 1H), 7.25 (m, 2H),7.28-7.40 (m, 18H), 9.79 (s, 1H).

Step C:2-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}[3-(α-D-mannopyranosyl)propyl]amino)ethylα-L-fucopyranoside

The title compound was prepared using procedures analogous to thosedescribed for ML-36, substituting3-(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl)-1-propanol for3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)propanol in Step E and2-aminoethyl (2,3,4-tri-O-acetyl-α-L-fucopyranoside) for 2-aminoethyl(3,4,6-tri-O-acetyl-2-(acetylamino)-2-deoxy-β-D-glucopyranoside) in stepF. UPLC Method B: m/e=623.3235 [M+1]; Rt=1.13 min.

Example 42

The synthesis of oligosaccharide linker 2,5-Dioxopyrrolidin-1-yl6-{[3-(α-L-fucopyranosyl)propyl][2-(α-D-mannopyranosyl)propyl]amino}hexanoate(ML-42) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-39, substituting3-(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl)-1-propanol for3-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)propanol in Step D. UPLCMethod B: m/e=621.3358 [M+1]; Rt=1.11 min.

Example 43

The synthesis of oligosaccharide linker 2,5-Dioxopyrrolidin-1-yl3-({6-[({bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}acetyl)amino]hexyl}amino)-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-D-alaninate(ML-43) having the following structure is described.

Step A: pentafluorophenyl6-[({bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}acetyl)amino]hexanoate

To a stirred solution of6-[({bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}acetyl)amino]hexanoicacid (1.0 g, 1.465 mmol, in DMF (7.7 mL) at 0° C. was added PFTU (627mg, 1.465 mmol) and, 5 min later, DIPEA (256μL, 1.465 mmol). The mixturewas allowed to warm up gradually to room temperature. After stirring for16 hr, the reaction mixture was concentrated and the residue waspurified by flash chromatography on silica gel (40 g), eluting with 300mL of EtOAc to wash out unpolar admixtures, and then with a mix solvent(v/v EtOAc/H₂O/MeOH/AcCN: 6/1/1/1), =6:1:1:1) to yield the titleproduct. LC-MS Method A: m/e=849.50 [M+1]; Rt=1.94 min.

Step B: 3-amino-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alaninehydrochloride

To a stirred solution of3-[(tert-butoxycarbonyl)amino]-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-D-alanine(1.0 g, 2.35 mmol) in CH₂Cl₂ (11.72 mL) was added HCl (4.0 M in dioxane,11.72 mL, 46.9 mmol). After stirring for 16 hours, the reaction mixturewas concentrated to give the title product, which was used withoutfurther purification. LC-MS Method A: m/e=327.19 [M+1]; Rt=1.73 min.

Step C:3-({6-[({bis[2-oxo-2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)ethyl]amino}acetyl)amino]hexanoyl}amino)-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alanine

To a stirred solution of pentafluorophenyl6-[({bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}acetyl)amino]hexanoate(270 mg, 0.318 mmol) in DMF at 0° C. was added3-amino-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alanine hydrochloride(303 mg, 0.306 mmol) and, 5 min later, DIPEA (111 μL, 0.636 mmol). Afterstirring at 0° C. for 2 hr, the reaction mixture was concentrated andthe residue was purified by flash chromatography on silica gel (22 g),eluting first with 100 mL of EtOAc and then 0-20% of Solvent B inSolvent A (Solvent A: v/v EtOAc/MeOH/AcCN/H₂O: 6/1/1/1; Solvent B v/vEtOAc/MeOH/AcCN/H₂O: 2/1/1/1) to afford the title compound. LC-MS MethodA: m/e=991.66 [M+1]; Rt=1.84 min.

Step D: 2,5-dioxopyrrolidin-1-yl3-({6-[({bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}acetyl)amino]hexyl}amino)-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-D-alaninate

The title compound was prepared using procedures analogous to thosedescribed for ML-1 substituting3-({6-[({bis[2-oxo-2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)ethyl]amino}acetyl)amino]hexanoyl}amino)-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alaninefor6-({2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}amino)-6-oxohexanoicacid in Step D. LC-MS Method A: m/e=1088.9 [M+1]; Rt=1.96 min.

Example 44

The synthesis of oligosaccharide linker 2,5-Dioxopyrrolidin-1-yl3-({6-[({bis[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}acetyl)amino]hexyl}amino)-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alaninate(ML-44) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-43 substituting3-[(tert-butoxycarbonyl)amino]-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alaninefor3-[(tert-butoxycarbonyl)amino]-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-D-alaninein Step B. LC-MS Method A: m/e=1088.9 [M+1]; Rt=1.96 min.

Example 45

The synthesis of oligosaccharide linker2,2′-{[2-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl]imino}bis(N-{[1-(α-D-mannopyranosyl)-1H-1,2,3-triazol-4-yl]methyl}acetamide)(ML-45) having the following structure is described.

Step A: benzyl6-[({bis[2-oxo-2-(prop-2-yn-1-ylamino)ethyl]amino}acetyl)amino]hexanoate

The title compound was prepared using the procedure analogous to thatdescribed for ML-1 Step B, substituting propargylamine for 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranoside.¹H NMR (CDCl₃, 400 MHz) δ 7.32 (m, 5H), 5.08 (s, 2H), 4.014 (s, 4H),3.298 (m, 4H), 3.23 (m, 2H), 3.21 (m, 2H), 2.32 (m, 2H), 2.22 (s, 2H),1.63 (m, 2H), 1.51 (m, 2H), 1.33 (m, 2H).

Step B: benzyl6-[({bis[2-oxo-2-({[(1-(α-D-mannopyranosyl)-1H-1,2,3-triazol-4-yl]methyl}amino)ethyl]amino}acetyl)amino]hexanoate

To a stirred solution of benzyl6-[({bis[2-oxo-2-(prop-2-yn-1-ylamino)ethyl]amino}acetyl)amino]hexanoate(546 mg, 1.165 mmol) and α-D-mannopyranosyl azide (598 mg, 2.91 mmol) inDMF (5.8 mL) was added DIPEA (1.0 mL, 5.83 mmol) and Cu(I) iodide (222mg, 1.165 mmol). The reaction mixture was allowed to stir at 60° C. for30 min until all CuI dissolved and then to stir at rt for 1 hr. Thereaction mixture was diluted by 10× volume of CH₃OH. The precipitatedinorganics was filtered out. The filtrate was concentrated and theresidue was purified by flash chromatography on silica gel (40 g),eluting with 0-60% Solvent B in Solvent A (Solvent A: v/vEtOAc/MeOH/AcCN/H₂O: 6/1/1/1; Solvent B v/v EtOAc/MeOH/AcCN/H₂O:2/1/1/1), to give the title compound. LC-MS Method A: m/e=879.31 [M+1];Rt=0.98 min.

Step C:2,2′-{[2-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl]imino}bis(N-{[1-(α-D-mannopyranosyl)-1H-1,2,3-triazol-4-yl]methyl}acetamide)

The title compound was prepared using procedures analogous to thosedescribed for ML-1, substituting benzyl6-[({bis[2-oxo-2-({[1-(α-D-mannopyranosyl)-1H-1,2,3-triazol-4-yl]methyl}amino)ethyl]amino}acetyl)amino]hexanoatefor benzyl 6-({2-[α-D-mannopyranosyl-(1→3[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}amino)-6-oxohexanoatein Step C. UPLC Method A: m/e=886.2 [M+1]; Rt=2.02 min.

Example 46

The synthesis of oligosaccharide linker2,2′-{[2-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl]imino}bis(N-{2-[(β-L-fucopyranosyl)oxy]ethyl}acetamide)(ML-46) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-7 substituting 2-aminoethyl β-L-fucopyranoside for2-aminoethyl α-L-fucopyranoside in Step B. UPLC Method B: m/e=780.361[M+1]; Rt=2.09 min.

Example 47

The synthesis of oligosaccharide linker6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-N-[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]-N-{2-[(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy}ethyl)amino]-2-oxoethyl}-6-oxohexanamide(ML-47) having the following structure is described.

The title compound was prepared using procedure analogous to thosedescribed for ML-29 substituting2,2′-{[6-(benzyloxy)-6-oxohexanoyl]imino}diacetic acid for2,2′-[(2-{[6-(benzyloxy)-6-oxohexyl]amino}-2-oxoethyl)imino]diaceticacid. UPLC Method B: m/e=1070.33 [M+1]; Rt=3.08 min.

Example 48

The synthesis of oligosaccharide linker2-{[2-({2-[(α-L-Fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl][2-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl]amino}-N-[2-(α-D-mannopyranosyloxy)ethyl]acetamide(ML-48) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-23 substituting 2-aminoethyl α-L-fucopyranoside for6-amino-N-(2-α-L-fucopyranosyl)ethyl)hexanamide in Step C and2-aminoethyl α-D-mannopyranoside for 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosidein Step D, respectively. UPLC Method B m/e=796.37 [M+1]; Rt=1.87 min.

Example 49

The synthesis of oligosaccharide linker2-{[2-({2-[(α-L-Fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl][2-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl]amino}-N-[2-(β-D-mannopyranosyloxy)ethyl]acetamide(ML-49) having the following structure is described.

Step A: benzyl{2-[(3,6-di-O-benzyol-β-D-galactopyranosyl)oxy]ethyl}carbamate

To a solution of benzyl2-({2-[(β-D-galactopyranosyl)oxy]ethyl}amino)carbamate (15.5 g, 43.4mmol, Beilstein J. Org. Chem. 2010, 6, 699) and 4 Å molecular sieves intoluene (150 mL) was added dibutylstannanone (23.4 g, 94 mmol). Afterrefluxing for 5 hr, the reaction mixture was slowly cooled down to 0°C., to which benzoyl chloride (11 mL, 95 mmol) was added dropwise. Theresulting mixture was allowed gradually warm up rt. After stirring at rtfor 24 hr, the reaction mixture was filtered and the filtrate wasconcentrated. The residue was purified by flash chromatography on silicagel (330 g), eluting with 0-75% EtOAc in hexanes, to give the titlecompound. TLC: silica gel, hexane/EtOAc: 1/1, R_(f)=0.35. UPLC Method B:calculated for C₃₀H₃₁NO₁₀ 565.19 observed m/e=566.21 [M+1]; Rt=1.87 min.¹H NMR (CDCl₃) δ 8.05-7.95 (4H, m), 7.60-7.25 (11H, m), 5.10-5.05 (1H,m), 5.02 (2H, s), 4.60-4.55 (1H, m), 4.50-4.45 (1H, m), 4.40-4.35 (1H,m), 4.20-4.15 (1H, m), 4.05-4.00 (1H, m), 3.90-3.80 (2H, m), 3.75-3.70(1H, m), 3.45-3.30 (1H, m), 3.35-3.25 (1H, m). Regiochemistry wasconfirmed by ¹H—¹³C one-bond correlation (HSQC); ¹H—¹³C multiple-bondcorrelation (HMBC); and ¹H—¹H NOE (NOESY) 2D NMR experiments.

Step B: benzyl{2-[(3,6-di-O-benoyl-β-D-mannopyranosyl)oxy]ethyl}carbamate

To a stirred solution of benzyl{2-[(3,6-di-O-benzyol-β-D-galactopyranosyl)oxy]ethyl}carbamate (1.17 g,2.069 mmol) in CH₂Cl₂ (26 mL) at −20° C. was added pyridine (2.4 mL,29.7 mmol) and, 5 min, triflic anhydride (1.1 mL, 6.51 mmol) dropwise.The mixture was allowed to slowly warm from −20° C. to 0° C. over 2 hr.The resulting mixture was diluted with CH₂Cl₂ (75 mL), which was washedwith cold 1.0 M HCl (100 mL), cold sat'd NaHCO₃ (100 mL), cold water(100 mL), and cold brine (100 mL). The organic phase was dried overNa₂SO₄ and concentrated at low temperature. The residue was dissolved inAcCN (20 mL), to which a solution of tetrabutylammonium nitrite (3.0 g,10.40 mmol) in AcCN (6 mL) was added. After stirring at 50° C. for 6 hr,the reaction mixture was concentrated and the residue was purified byflash column chromatography on silica gel (330 g), eluting with 0-75%EtOAc in hexanes, to give the title compound. TLC: silica gel,hexane/Ethylacetate: 1/1, R_(f)=0.33. UPLC Method B: calculated forC₃₀H₃₁NO₁₀ 565.19 observed m/e=566.22 [M+1]; Rt=1.84 min. ¹H NMR (CDCl₃)δ 8.10-8.00 (4H, m), 7.55-7.25 (11H, m), 5.05-5.00 (3H, m), 4.72-4.68(1H, m), 4.64-4.58 (2H, m), 4.24-4.20 (1H, m), 4.17-4.12 (1H, m),3.93-3.87 (1H, m), 3.77-3.72 (1H, m), 3.65-3.60 (1H, m), 3.46-3.34 (2H,m). Stereochemistry was confirmed by 1H-13C one-bond correlation (HSQC);1H-13C multiple-bond correlation (HMBC); and 1H-1H NOE (NOESY) 2D NMRexperiments.

Step C: benzyl[2-(β-D-mannopyranosyloxy)ethyl]carbamate

To a stirred solution of benzyl{2-[(3,6-di-O-benzoyl-β-D-fucopyranosyl)oxy]ethyl}carbamate (287 mg,0.507 mmol) in CH₃OH (5 mL) was added NaOCH (0.1 mL, 0.05 mmol, 0.5 M inCH₃OH). After 4 hr, Amberlite IR 120 (H) ion exchange resin (pre-washedwith CH₃OH 3×5 mL) was added to the stirred reaction mixture. After 15min, the resin was filtered off and washed with CH₃OH (3×5 mL). Thefiltrate was concentrated and the residue was purified by flashchromatography on (50 g) on C18 reverse phase silica gel, eluting with5-60% AcCN in H₂O, to give the title compound. UPLC Method B: calculatedfor C₁₆H₂₃NO₈ 357.14 observed m/e=358.16 [M+1]; Rt=1.40 min. ¹H NMR(CD₃OD) δ 7.35-7.24 (5H, m), 5.04 (2H, s), 4.50-4.48 (1H, m), 3.90-3.82(3H, m), 3.74-3.60 (2H, m), 3.55-3.50 (1H, m), 3.42-3.37 (1H, m),3.36-3.30 (2H, m), 3.18-3.12 (1H, m).

Step D: 2-aminoethyl β-D-mannopyranoside

A mixture of benzyl[2-(β-D-mannopyranosyloxy)ethyl]carbamate (133 mg,0.372 mmol), and Pd/C (20 mg, 0.019 mmol) in water (5 mL) was allowed tostir under a balloon of H₂ at rt for 4 hr. The catalyst was filtered offand washed with H₂O (3×5 mL). The fitrate was concentrated to give thetitle compound. ¹H NMR (CD₃OD) δ 4.53-4.52 (1H, m), 3.93-3.85 (3H, m),3.72-3.64 (2H, m), 3.53-3.49 (1H, m), 3.44-3.42 (1H, m), 3.22-3.18 (1H,m), 2.92-2.85 (2H, m).

Step E:2-{[2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl][2-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl]amino}-N-[2-(β-D-mannopyranosyloxy)ethyl]acetamide

The title compound was prepared using procedures analogous to thosedescribed for ML-48 substituting 2-aminoethyl β-D-mannopyranoside for2-aminoethyl α-D-mannopyranoside. UPLC Method B: m/e=796.37 [M+1];Rt=2.19 min.

Example 50

The synthesis of oligosaccharide linker2-{[2-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl][2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}-N-(2-{1[α-D-mannopyranosyl],4[α-D-mannopyranosyl]-oxy}butyl)acetamide(ML-50) having the following structure is described.

Step A: Benzyl(1[2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl],4[2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl]-dihydroxybutan-2-yl)carbamate

To a 500 mL rb flask containing 4 Å Molecular sieves, was added asolution of (S)-benzyl (1,4-dihydroxybutan-2-yl)carbamate (2.7 g, 11.28mmol), and 2,3,4,6-tetra-O-benzoyl-D-mannopyranosyl trichloroacetimidate(17.35 g, 23.42 mmol, prepared according to Organic Letters, 2003, vol.5, No. 22, 4041) in anhydrous Dichloromethane (200 mL). The mixture wascooled down to −30° C. and trimethylsilyl trifluoromethanesulfonate (0.4mL, 2.214 mmol) was added. After stirring at −30 to ambient temperaturefor 4 h under nitrogen, reaction mixture was quenched with TEA (3.15 mL,22.57 mmol), filtered and concentrated under reduce pressure. Theresidue was purified by silica chromatography (0-60% EtOAc/hexanes) togive the title compound. (TLC: silica gel, Hexane/Ethylacetate:3/2,product R_(f)=0.4). ¹H NMR (Chloroform-d, 500 MHz): δ 8.15-8.10 (4H, m),8.10-8.05 (4H, m), 8.00-7.95 (4H, m), 7.80-7.75 (4H, m), 7.65-7.55 (4H,m), 7.45-7.20 (15H, m), 6.15-6.10 (2H, m), 5.95-5.85 (2H, m), 5.80-5.75(2H, m), 5.15-5.10 (4H, m), 4.75-4.65 (2H, m), 4.55-4.40 (4H, m),4.25-4.20 (1H, m), 4.10-4.00 (2H, m), 3.75-3.65 (2H, m), 2.15-2.05 (2H,m).

Step B: Benzyl(1[α-D-mannopyranosyl],4[α-D-mannopyranosyl]-dihydroxybutan-2-yl)carbamate

To a solution of Benzyl(1[2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl],4[2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl]-dihydroxybutan-2-yl)carbamate(9.72 g, 6.96 mmol) in Methanol (30 mL) was added 0.5M sodium methoxide(1.5 mL, 0.750 mmol) in methanol. After stirring at room temperature for48 hour, amberlite IR 120 (H) ion exchange resin (pre-washed withmethanol 3×30 ml) was added to reaction mixture, and allowed stirringfor additional 15 minutes. The ion exchange resin was filtered off andwashed with methanol (2×5 mL). The filtrate was concentrated down andthe residue was purified on 86 g C18 column reverse phase, eluting with5% CH₃CN in water (3 cv) then 5% to 50% CH₃CN in water (20 cv) to givethe title compound as a white solid. UPLC-MS: calculated for C₂₄H₃₇NO₁₄563.22 observed m/e: 564.24 (M+H)+(Rt: 2.31/5.00 min).

Step C:1(α-D-mannopyranosyl),4(α-D-mannopyranosyl)-dihydroxybutan-2-amine

A mixture of Benzyl(1[α-D-mannopyranosyl],4[α-D-mannopyranosyl]-dihydroxybutan-2-yl)carbamate(3.73 g, 6.62 mmol), and Pd/C (0.300 g, 0.282 mmol) in water (60 mL) wasallowed to stir under a balloon of H₂ at rt for 2 h. The catalyst wasfiltered off and washed with H₂O (3×10 mL). The filtrate wasconcentrated to give the title compound. UPLC-MS: calculated forC₁₆H₃₁NO₁₂ 429.18 observed m/e: 430.21 (M+H)+(Rt: 1.37/5.00 min).

Step D:2-{[2-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl][2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}-N-(2-{1-[α-D-mannopyranosyl]4[-α-D-mannopyranosyl]-oxy}butyl)acetamide

The title compound was prepared using procedure analogous to thosedescribed for ML-29 substituting1(α-D-mannopyranosyl),4(α-D-mannopyranosyl)-dihydroxybutan-2-amine for2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranoside.UPLC Method B: calculated for C₄₀H₆₂N₅O₂₄ 1001.42, observed m/e: 1002.48[M+1]; Rt=2.05 min.

Example 51

The synthesis of oligosaccharide linker2-{[2-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl][2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-13-D-mannopyranosyl]oxy}ethyl)acetamide(ML-51) having the following structure is described.

Step A: (2-((3,6-O-benzoyl-β-D-galactopyranosyl)oxy)chloroethyl)

To a 500 mL Rb flask containing 4 Å Molecular sieves,2-chloroethyl-3-D-galactopyranoside (6.58 g, 27.1 mmol, preparedaccording to Carbohydr. Res. 1992, 223, 303), dibutylstannanone (14.5 g,58.2 mmol) and anhydrous Toluene (150 mL) were added. The mixture wasstirred at reflux for 3 h. After 3 h, the reaction mixture were cooleddown to room temperature and then to 0° C. To a cold solution of thereaction crude at 0° C., benzoyl chloride (6.7 ml, 57.7 mmol) was added,and the resulting mixture was stirred at 0° C. to ambient temperature.After 24 hours, the reaction mixture was filtered off and filtrate wasconcentrated under reduce pressure. The residue was purified by silicachromatography (0-75% EtOAc/hexanes) to give the title compound.UPLC-MS: calculated for C₂₂H₂₃ClO₈ 450.11 observed m/e: 473.11(M+Na)+(Rt: 1.82/5.00 min). ¹H NMR (Chloroform-d, 500 MHz): δ 8.10-8.05(2H, m), 8.05-8.00 (2H, m), 7.60-7.55 (2H, m), 7.50-7.40 (4H, m),5.20-5.10 (1H, m), 4.70-4.60 (1H, m), 4.55-4.50 (1H, m), 4.50-4.45 (1H,m), 4.25-4.20 (1H, m), 4.20-4.15 (1H, m), 4.12-4.08 (1H, m), 4.00-3.95(1H, m), 3.90-3.85 (1H, m), 3.72-3.68 (2H, m). Regiochemistry wasconfirmed by ¹H—¹³C one-bond correlation (HSQC); ¹H—¹³C multiple-bondcorrelation (HMBC); and ¹H—¹H NOE (NOESY) 2D NMR experiments.

Step B: (2-((3,6-O-benzoyl-β-D-mannopyranosyl)oxy)chloroethyl)

To a solution of(2-((3,6-O-benzoyl-β-D-galactopyranosyl)oxy)chloroethyl) (1 g, 2.218mmol) in DCM (28 mL) at −20° C. was added pyridine (2.5 mL, 30.9 mmol).After stirring 5 minutes, TriflicAnhydride (1.2 mL, 7.10 mmol) was addeddropwise, and the mixture was stirred while allowing to warm from −20°C. to 0° C. over 2 hours. The resulting mixture was diluted with 75 mlDCM and washed with (1×100 mL) of cold 1 M HCl; (1×100 mL) of coldaqueous NaHCO₃; (1×100 mL) of cold water and 100 ml of cold brine. Theorganic phase was dried over Na₂SO₄ and concentrated in vacuo at lowtemperature. The residue was used directly in the next step withoutfurther purification. Solution of tetrabutylammonium nitrite (3.3 g,11.44 mmol) in 5 ml of anhydrous acetonitrile was added to the solutionof triflated intermediate in dry acetonitrile (22 mL) and then allowedto react at 50° C. for 5 hours. The resulting mixture was concentratedunder reduce pressure and directly purified by silica chromatography(0-75% EtOAc/hexanes) to give the title compound. UPLC-MS: calculatedfor C₂₂H₂₃ClO₈ 450.11 observed m/e: 451.13 (M+H)+(Rt: 1.80/5.00 min)¹HNMR (Chloroform-d, 500 MHz): δ 8.15-8.05 (4H, m), 7.65-7.60 (2H, m),7.50-7.40 (4H, m), 5.12-5.08 (1H, m), 4.77-4.72 (2H, m), 4.68-4.65 (1H,m), 4.34-4.32 (1H, m), 4.24-4.14 (2H, m), 3.90-3.84 (1H, m), 3.71-3.66(3H, m), 2.98-2.96 (1H, b), 2.41-2.39 (1H, b). Stereochemistry wasconfirmed by ¹H—¹³C one-bond correlation (HSQC); ¹H—¹³C multiple-bondcorrelation (HMBC); and ¹H—¹H NOE (NOESY) 2D NMR experiments.

Step C:(2-((2,4-O-benzyl,3,6-O-benzoyl-β-D-mannopyranosyl)oxy)chloroethyl)

In a 250 mL rb flask, 2-(benzyloxy)-1-methylpyridin-1-iumtrifluoromethanesulfonate (5 g, 14.31 mmol), magnesium oxide (0.085 g,2.118 mmol), trifluorotoluene (40 mL), and(2((3,6-O-benzoyl-β-D-mannopyranosyl)oxy)chloroethyl) (1.91 g, 4.24mmol) were added. The heterogeneous reaction mixture is then stirred at82° C. for 48 h. Upon reaction completion, reaction mixture wasfiltrated and concentrated under reduce pressure. The residue waspurified by silica chromatography (0 to 75% EtOAc/hexanes) to give thetitle compound. UPLC-MS: calculated for C₃₆H₃₅ClO₈ 630.20 observed m/e:631.21 (M+H)+(Rt: 3.97/5.00 min).

Step D: (2-((2,4-O-benzyl-β-D-mannopyranosyl)oxy)chlomethyl)

To a solution of(2-((2,4-O-benzyl,3,6-O-benzoyl-β-D-mannopyranosyl)oxy)chloroethyl),(1.6 g, 2.54 mmol) in methanol (25 mL) and DCM (5 mL) was added 0.5Msodium methoxide (0.5 mL, 0.25 mmol) in methanol. After stirring at roomtemperature for 4 hour, amberlite IR 120 (H) ion exchange resin(pre-washed with methanol 3×5 mL) was added to reaction mixture, andallowed stirring for additional 15 minutes. The ion exchange resin wasfiltered off and washed with methanol (3×5 mL). The filtrate wasconcentrated under reduce pressure and the residue was purified bysilica chromatography (0-75% EtOAc/hexanes) to give the title compound.UPLC-MS: calculated for C₂₂H₂₇ClO₆ 422.15 observed m/e: 423.14(M+H)+(Rt: 1.90/5.00 min).

Step E: (2-((2,4-O-benzyl-β-D-mannopyranosyl)oxy)azidoethyl)

To a solution of (2-((2,4-O-benzyl-β-D-mannopyranosyl)oxy)chloroethyl)(800 mg, 1.892 mmol) in anhydrous DMF (25 mL) was added sodium azide(140 mg, 2.154 mmol) at room temperature. The reaction mixture was thenheated to 70° C. and stirred for 16 h under nitrogen. Upon reactioncompletion, crude reaction mixture was cooled down to room temperatureand poured onto ice water (200 mL) and extracted with ether (3×100 mL).The organic layers were combined and washed with brine (2×100 mL), driedover Na₂SO₄, filtered and concentrated under reduce pressure. Theresidue was purified by silica chromatography (0-100% EtOAc/hexanes) togive the title compound. . UPLC-MS: calculated for C₂₂H₂₇N₃O₆ 429.19observed m/e: 430.19 (M+H)+(Rt: 1.87/5.00 min).

Step F:(2-((2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl-(1→6)]-(2,4-O-benzyl-β-D-mannopyranosyl)oxy)azidoethyl)

To a 250 mL Rb flask containing 4 Å Molecular sieves was added asolution of (2-((2,4-O-benzyl-β-D-mannopyranosyl)oxy)azidoethyl) (710mg, 1.653 mmol) and 2,3,4,6-tetra-O-benzoyl-D-mannopyranosyltrichloroacetimidate (2.6 g, 3.51 mmol, prepared according to OrganicLetters, 2003, vol. 5, No. 22, 4041) in anhydrous Dichloromethane (30mL). The mixture was cooled down to −30° C. and trimethylsilyltrifluoromethanesulfonate (0.03 mL, 0.166 mmol) was added. Afterstirring at -30 to ambient temperature for 4 h under nitrogen, reactionmixture was quenched with TEA (0.1 mL, 0.717 mmol), filtered andconcentrated under reduce pressure. The residue was purified by silicachromatography (0-75% EtOAc/hexanes) to give the title compound. (TLC:silica gel, Hexane/Ethylacetate:3/2, product R_(f)=0.6). ¹H NMR(Chloroform-d, 500 MHz): δ 8.15-7.80 (16H, m), 7.62-7.49 (8H, m),7.47-7.32 (20H, m), 7.30-7.15 (6H, m), 6.13-6.05 (2H, m), 6.01-5.91 (2H,m), 5.87-5.85 (1H, m), 5.71-5.69 (1H, m), 5.43-5.42 (1H, m), 5.26-5.23(1H, m), 5.13-5.12 (1H, m), 5.03-5.00 (1H, m), 4.84-4.81 (1H, m),4.68-4.63 (2H, m), 4.60-4.55 (2H, m), 4.54-4.48 (2H, m), 4.31-4.25 (2H,m), 4.20-4.15 (1H, m), 4.08-4.07 (1H, m), 3.98-3.88 (3H, m), 3.84-3.80(1H, m), 3.78-3.73 (1H, m), 3.62-3.54 (2H, m), 3.38-3.33 (1H, m).

Step G:(2-((α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-(2,4-O-benzyl,β-D-mannopyranosyl)oxy)azidoethyl)

To a solution of2-((2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl-(1→6)]-(2,4-O-benzyl-β-D-mannopyranosyl)oxy)azidoethyl(2.52 g, 1.588 mmol) in Methanol (20 mL) and DCM (4 mL) was added 0.5Msodium methoxide (0.32 mL, 0.16 mmol) in methanol. After stirring atroom temperature for 24 hour, amberlite IR 120 (H) ion exchange resin(pre-washed with methanol 3×30 mL) was added to reaction mixture, andallowed stirring for additional 15 minutes. The ion exchange resin wasfiltered off and washed with methanol (3×5 mL). The filtrate wasconcentrated under reduce pressure and the residue was purified byreverse phase chromatography (C18 column) (ACN/H₂O with no modifier) toafford the title product. UPLC-MS: calculated for C₃₄H₄₂N₃O₁₆ 753.30observed m/e: 754.29 (M+H)+(Rt: 2.93/5.00 min).

Step H: 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-β-D-mannopyranoside

A mixture of2-((α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-(2,4-O-benzyl,13-D-mannopyranosyl)oxy)azidoethyl (1.05 g, 1.393 mmol), and Pd/C (0.148g, 0.139 mmol) in water (25 mL) was allowed to stir under a balloon ofH₂ at rt for 16 h. The catalyst was filtered off and washed with H₂O(3×5 mL). The filtrate was concentrated under reduce pressure to givethe title compound. UPLC-MS: calculated for C₂₀H₃₂NO₁₆ 547.21 observedm/e: 548.23 (M+H)+(Rt: 1.07/5.00 min)¹H NMR (D₂O, 500 MHz): δ 5.06-5.05(1H, m), 4.86-4.85 (1H, m), 4.655-4.65 (1H, m), 4.13-4.12 (1H, m),4.02-4.01 (1H, m), 3.95-3.82 (6H, m), 3.80-3.67 (8H, m), 3.66-3.57 (3H,m), 3.53-3.49 (1H, m), 2.90-2.85(2H, m).

Step I:2-{[2-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl][2-({2-[(α-L-fucopyranosyl)oxy]ethyl}amino)-2-oxoethyl]amino}-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-β-D-mannopyranosyl]oxy}ethyl)acetamide

The title compound was prepared using procedure analogous to thosedescribed for ML-29 substituting 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-β-D-mannopyranosidefor 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranoside.UPLC Method B: calculated for C₄₄H₇₃N₅O₂₈ 1119.44, observed m/e: 1120.46[M+1]; Rt=2.09 min.

Example 52

The synthesis of oligosaccharide linker6-[(2,5-dioxopyrrolidin-1-yl)oxy]-N-{2-[(β-D-mannopyranosyl)oxy]ethyl}-6-oxohexanamide(ML-52) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-1 substituting 2-aminoethyl β-D-mannopyranoside for2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosidein step B. UPLC-MS: calculated for C₁₈H₂₈N₂O₁₁ 448.17 observed m/e:449.19 (M+H)+(Rt: 1.96/5.00 min).

Example 53

The synthesis of oligosaccharide linker4-[(2,5-dioxopyrrolidin-1-yl)oxy]-N-{2-[(α-L-fucopyranosyl)oxy]ethyl}4-oxobutanamide(ML-53) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-4 substituting 4-(benzyloxy)-4-oxobutanoic acid for6-(benzyloxy)-6-oxohexanoic acid in Step A. UPLC-MS: calculated forC₁₆H₂₄N₂O₁₀ 404.14 observed m/e: 405.14 (M+H)+(Rt: 1.87/5.00 min).

Example 54

The synthesis of oligosaccharide linker2,2′-{[2-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl]imino}bis[N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-β-D-glucopyranosyl]oxy}ethyl)acetamide](ML-54) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-15 substituting 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-β-D-glucopyranosidefor 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosidein Step B. UPLC Method B: calculated for C₅₆H₉₃N₅O₃₉ 1459.54, observedm/e: m/e=1460.62 [M+1]; Rt=0.92 min.

Example 55

The synthesis of oligosaccharide linker2-(2-{([α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy)ethyl}{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)ethylα-L-fucopyranoside (ML-55) having the following structure is described.

Step A: 2-aminoethyl2,3,4,6-penta-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-penta-benzoyl-α-D-mannopyranosyl-(1→6)]-2,4-dibenzoyl-α-D-mannopyranoside

To a nitrogen flushed solution of 2-azidoethyl2,3,4,6-penta-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-O-penta-benzoyl-α-D-mannopyranosyl-(1→6)]-2,4-di-O-benzoyl-α-D-mannopyranoside(25 g, 15.5 mmol WO 2010/088294 A1) in EtOAc (300 mL) was added 10%Palladium on Carbon (1.65 g) and the resulting mixture stirred at roomtemperature under a balloon of hydrogen overnight. Mixture filteredthrough Celite and the filtrate evaporated, the residue was purified bysilica gel column chromatography (Teledyne Isco: 330 g) eluent: gradient2-5% MeOH in DCM over 8CV to give the title compound (18 g, 73%) as anoff-white foam. UPLC Method C: calculated for C₉₀H₇₇NO₂₆ 1587.47,observed m/e: 1588.6636 [M+1]; Rt=4.17 min.

Step B: benzyl6-(2-{2,3,4,6-penta-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-penta-O-benzoyl-α-D-mannopyranosyl-(1→6]-2,4-di-O-benzoyl-α-D-mannopyranosyl]-2-oxyethyl}amino)hexanoate

To a solution of 2-aminoethyl2,3,4,6-penta-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-penta-O-benzoyl-α-D-mannopyranosyl-(1→6)]-2,4-di-O-benzoyl-α-D-mannopyranoside(18 g, 11.35 mmol) and benzyl 6-oxohexanoate (1 g, 4.54 mmol) in DCM(150 mL) was added acetic acid (0.26 mL, 4.54 mmol) and sodiumtriacetoxyborohydride (2.41 g, 11.35 mmol) and the resulting mixturestirred at room temperature overnight. Mixture evaporated and theresidue dissolved in EtOAc (300 mL) and washed with sat. NaHCO₃ (2×300mL), sat. NaCl (200 mL), dried over Na₂SO₄, filtered and evaporated. Theresidue was purified by silica gel column chromatography (Teledyne Isco:2×330 g) eluent: gradient 2-5% MeOH in DCM over 8CV to give the titlecompound (4.8 g, 59%) as a white foam. ¹H NMR (CDCl₃) δ 8.33 (2H, m),8.18 (2H, m), 8.13 (2H, dd, J=8.0 and 1.4 Hz), 8.10 (2H, dd, J=8.0 and1.4 Hz), 8.06 (2H, m), 8.05 (2H, dd, J 4.8 and 1.6 Hz), 7.84 (4H, m),7.78 (2H, dd, J=8.0 and 1.4 Hz), 7.74 (2H, dd, J=8.0 and 1.4 Hz),7.67-7.48 (8H, m), 7.47-7.30 (23H, m), 7.25 (2H, t, J=7.8 Hz), 6.14 (1H,t, J=10.1 Hz), 6.10 (1H, t, J=10.0 Hz), 6.03 (1H, dd, J=10.1 and 3.3Hz), 5.93 (1H, t, J=10.0 Hz), 5.80 (2H, m), 5.75 (1H, dd, J=10.1 and 3.3Hz), 5.42 (1H, d, J=1.9 Hz), 5.38 (1H, dd, J=3.3 and 1.9 Hz), 5.20 (1H,s), 5.18 (1H, d, J=1.8 Hz), 5.11 (2H, s), 4.69 (1H, dd, J=9.7 and 3.5Hz), 4.67 (1H, dd, J=12.4 and 2.6 Hz), 4.62 (1H, dd, J=12.2 and 2.4 Hz),4.57 (1H, m), 4.48 (1H, dt, J=10.1 and 2.8 Hz), 4.42-4.31 (3H, m), 4.22(1H, dd, J=10.8 and 6.3 Hz), 4.09 (1H, dt, J=10.0 and 5.4 Hz), 3.83 (1H,d, J=10.6 Hz), 3.78 (1H, m), 3.02 (2H, m), 2.73 (2H, t, J=7.3 Hz), 2.37(2H, t, J=7.6 Hz), 1.71 (2H, m), 1.59 (2H, m), 1.40 (2H, m).

Step C: 2-oxoethyl 2,3,4-tri-O-acetyl-α-L-fucopyranoside

To a solution of prop-2-en-1-yl 2,3,4-tri-O-acetyl-α-L-fucopyranoside(1.34 g, 4.06 mmol) in acetone (30 mL) and water (7.5 mL) was added4-methylmorpholine 4-oxide (950 mg, 8.11 mmol) followed by the additionof 2.5% OsO₄ in tert-butanol (2.04 mL, 0.162 mmol). The mixture wasallowed to stir at rt for 16 hr. To the resulting mixture was then addeda solution of NaIO₄ (1.74 g, 8.11 mmol) in water (15 mL). After stirringfor additional 4 hr, the precipitate was filtered and washed withacetone (50 mL). The volume of the filtrate was reduced to approximately⅓ of the initial volume and then diluted with sat. NaHCO₃ (100 mL). Themixture was extracted with EtOAc (3×50 mL). The organic phases werecombined and washed with brine, dried over Na₂SO₄, and concentrated. Theresidue was purified by silica gel column chromatography (Teledyne Isco:120 g), eluting with 0-80% EtOAc in hexanes to give the title compound(660 mg, 49%). ¹H NMR (CDCl₃) δ 9.72 (1H, s), 5.44 (1H, dd, J=10.9 and3.3 Hz), 5.35 (1H, dd, J=3.4 and 1.8 Hz), 5.19 (1H, dd, J=10.9 and 3.8Hz), 5.13 (1H, d, J=3.8 Hz), 4.26 (3H, m), 2.19 (3H, s), 2.15 (3H, s),2.02 (3H, s), 1.17 (3H, d, J=6.6 Hz).

Step D: benzyl6-{[{2-(2,3,4-tri-O-acetyl-α-L-fucopyranosyl)-oxy}ethyl](2-{(2-{2,3,4,6-penta-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-penta-O-benzoyl-α-D-mannopyranosyl-(1→6)]-2,4-di-O-benzoyl-α-D-mannopyranosyl]-oxy}ethyl)amino}hexanoate

To a solution of benzyl6-(2-{2,3,4,6-penta-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-penta-O-benzoyl-α-D-mannopyranosyl-(1→6)]-2,4-di-O-benzoyl-α-D-mannopyranosyl]-2-oxyethyl}amino)hexanoate (1.3 g, 0.725 mmol) and 2-oxoethyl2,3,4-tri-O-acetyl-α-L-fucopyranoside (651 mg, 1.96 mmol) in DCM (20 mL)was added acetic acid (0.042 mL, 0.725 mmol) and sodium triacetoxyborohydride (307 mg, 1.45 mmol) and the resulting mixture stirred atroom temperature overnight. Mixture evaporated and the residuepartitioned between EtOAc (60 mL) and sat. NaHCO₃ (80 mL); organic layerwashed with a further portion of sat. NaHCO₃ (80 mL), sat. NaCl (50 mL),dried over Na₂SO₄, filtered and evaporated. The residue purified bysilica gel column chromatography (Teledyne Isco: 80 g) eluent: gradient20-100% EtOAc in Hexanes over 10CV to give the title compound (1.5 g,99%) as a white foam. ¹H NMR (CDCl₃) δ 8.34 (2H, dd, J 6.7 and 3.2 Hz),8.15 (2H, m), 8.10 (4H, m), 8.07 (2H, dd, J=8.0 and 1.4 Hz), 8.05 (2H,dd, J 8.1 and 1.5 Hz), 7.88 (4H, m), 7.78 (2H, dd, J=8.0 and 1.4 Hz),7.72 (2H, dd, J=7.8 and 1.5 Hz), 7.62-7.55 (6H, m), 7.54-7.36 (14H, m),7.34-7.29 (11H, m), 7.25 (2H, t, J=7.7 Hz), 6.14 (1H, t, J=10.0 Hz),6.07 (1H, m), 6.03 (2H, m), 5.83 (1H, dd, J=3.3 and 1.8 Hz), 5.75 (2H,m), 5.41 (1H, m), 5.39 (2H, m), 5.35 (1H, d, J=3.8 Hz), 5.33 (2H, m),5.59 (1H, d, J=3.7 Hz), 5.18 (3H, m), 5.16 (1H, d, J=3.8 Hz), 5.13 (1H,t, J=4.2 Hz), 5.10 (1H, d, J=3.7 Hz), 5.06 (2H, s), 4.62 (2H, m), 4.54(1H, m), 4.50 (2H, m), 4.57 (1H, m), 4.33 (3H, m), 4.25 (2H, m), 4.19(2H, m), 3.80 (2H, m), 2.82 (2H, m), 2.60 (1H, t, J=7.3 Hz), 2.32 (2H,t, J=7.5 Hz), 2.20 (3H, s) 2.11 (3H, s) 2.02 (3H, s), 1.63 (2H, m), 1.51(1H, m) 1.34 (1H, m) 1.18 (3H, d, J=6.6 Hz).

Step E: methyl6{[{2-(-α-L-fucopyranosyl)-oxy}ethyl](2-{(2-{-α-D-mannopyranosyl-(1→3)-[-α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]-oxy}ethyl)amino}hexanoate

To a solution of benzyl6-{[{2-(2,3,4-tri-O-acetyl-α-L-fucopyranosyl)-oxy}ethyl](2-{(2-{2,3,4,6-penta-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-penta-O-benzoyl-α-D-mannopyranosyl-(1→6)]-2,4-di-O-benzoyl-α-D-mannopyranosyl]-oxy}ethyl)amino}hexanoate(1.5 g, 0.71 mmol) in a mixture of DCM (5 mL) and MeOH (15 mL) was addedsodium methoxide (0.284 mL of a 0.5M soln in MeOH, 0.142 mmol) and themixture stirred at room temperature for 4 days. Mixture evaporated to avolume of ˜4 mL and added dropwise to stirred acetonitrile (80 mL) togive a white precipitate. Mixture centrifuged at 3500 rpm for 20 mins,supernatant decanted and solids re-suspended in acetonitrile (80 mL) andcentrifuged at 3500 rpm for a further 20 mins, supernatant decanted andsolids dried under a stream of dry nitrogen to give the title compound(580 mg, 94%) as a white solid. UPLC Method B: calculated for C₃₅H₆₃NO₂₃865.38, observed m/e: 866.48 [M+1]; Rt=1.71 min.

Step F:6-{[{2-(-α-L-fucopyranosyl)-oxy}ethyl](2-{(2-{-α-D-mannopyranosyl-(1→3)-[-α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]-oxy}ethyl)amino}hexanoicacid

To a solution of methyl6-{[{2-(-α-L-fucopyranosyl)-oxy}ethyl](2-{(2-{-α-D-mannopyranosyl-(1→3)-[-α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]-oxy}ethyl)amino}hexanoate(580 mg, 0.67 mL) in water (3 mL) was added 5N NaOH (0.161 mL, 0.804mmol) and the resulting mixture stirred at room temperature for 6 hours.Acetic acid (0.039 mL, 0.683 mmol) added and mixture lyophilized to givethe title compound (620 mg, 100%). UPLC Method B: calculated forC₃₄H₆₁NO₂₃ 851.36, observed m/e: 852.48 [M+1]; Rt=1.74 min.

Step G:2-(2-{([α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy)ethyl}{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)ethylα-L-fucopyranoside

To a suspension of6-{[{2-(-α-L-fucopyranosyl)-oxy}ethyl](2-{(2-{-α-D-mannopyranosyl-(1→3)-[-α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]-oxy}ethyl)amino}hexanoicacid (100 mg, 0.117 mmol) in anhydrous DMF (2 mL) was added Hunig's base(0.082 mL, 0.47 mmol) and1-(((2,5-dioxopyrrolidin-1-yl)oxy)(pyrrolidin-1-yl)methylene)pyrrolidin-1-iumhexafluorophosphate(V) (58 mg, 0.141 mmol) and the resulting mixturestirred at room temperature for 30 mins. TFA (0.036 mL, 0.47 mmol) addedand the resulting mixture added dropwise to anhydrous acetonitrile (40mL) to form a white precipitate. Mixture centrifuged at 3500 rpm for 20mins, solvent decanted and solid re-suspended in acetonitrile (40 mL)and centrifuged at 3500 rpm for 20 mins Solvent decanted and solid driedunder a stream of dry nitrogen to give the title compound (84 mg, 75%).UPLC Method B: calculated for C₃₈H₆₄N₂O₂₅ 948.38, observed m/e: 949.48[M+1]; Rt=3.64 min.

Example 56

The synthesis of oligosaccharide linker2-(2-{([α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy)ethyl}{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)ethylβ-L-fucopyranoside (ML-56) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-55 substituting prop-2-en-1-yl2,3,4-tri-O-acetyl-β-L-fucopyranoside for prop-2-en-1-yl2,3,4-tri-O-acetyl-α-L-fucopyranoside in Step C. UPLC Method B:m/e=949.48 [M+1]; Rt=3.70 min.

Example 57

The synthesis of oligosaccharide linker3-(2-{([α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy)ethyl}{6-(2,5-dioxopyrrolidin-1-yl)-6-oxohexyl}amino)propylα-L-fucopyranoside (ML-57) having the following structure is described.

Step A: benzyl6-{[3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)propyl](2-[2-{2,3,4,6-penta-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-penta-O-benzoyl-α-D-mannopyranosyl-(1→6)]-2,4-di-O-benzoyl-α-D-mannopyranosyl}-oxy]ethyl)amino}hexanoate

Prepared from benzyl6-(2-{2,3,4,6-penta-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-penta-O-benzoyl-α-D-mannopyranosyl-(1→6)]-2,4-di-O-benzoyl-α-D-mannopyranosyl]-2-oxyethyl}amino)hexanoate [ML-55 step B] and3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)propanal [ML-36 Step E]according to the procedure outlined for ML-55 Step D. ¹H NMR (CDCl₃) δ8.35 (2H, dd, J=6.4 and 2.9 Hz), 8.17 (2H, d, J=7.7 Hz), 8.10 (4H, m),8.06 (2H, dd, J=7.1 and 1.5 Hz), 8.04 (2H, dd, J=7.7 and 1.5 Hz), 7.89(2H, m), 7.87 (2H, m), 7.80 (2H, dd, J=7.9 and 1.4 Hz), 7.74 (2H, m),7.61 (2H, m), 7.59-7.55 (4H, m), 7.49 (2H, d, J=7.2 Hz), 7.45-7.35 (12H,m), 7.35-7.27 (26H, m), 7.23 (2H, m), 6.18 (1H, t, J=9.9 Hz), 6.11-6.02(3H, m), 5.85 (1H, dd, J=3.3 and 1.8 Hz), 5.75 (2H, m), 5.37 (2H, m),5.17 (2H, m), 5.06 (2H, s), 4.77 (1H, d, J=12.0 Hz), 4.70 (2H, d, J=8.3Hz), 4.67-4.60 (5H, m), 4.56 (2H, d, J=8.6 Hz), 4.55-4.47 (4H, m), 4.33(3H, m), 4.19 (1H, dd, J=11.0 and 4.8 Hz), 4.01 (1H, dd, J=10.5 and 4.7Hz), 3.86 (1H, m), 3.79 (1H, d, J=11.2 Hz), 3.75 (2H, m), 3.62 (1H, m),2.79 (2H, t, J=6.5 Hz), 2.52 (4H, m), 2.32 (2H, t, J=7.6 Hz), 1.65 (4H,m), 1.48 (2H, m), 1.30 (3H, m), 1.25 (3H, d, J=6.6 Hz).

Step B: methyl6-{[3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)propyl](2-[2-{-α-D-mannopyranosyl-(1→3)-[-α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl}-oxy]ethyl)amino}hexanoate

To a solution of benzyl6-{[3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)propyl](2-[2-{2,3,4,6-penta-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-penta-O-benzoyl-α-D-mannopyranosyl-(1→6)]-2,4-di-O-benzoyl-α-D-mannopyranosyl}-oxy]ethyl)amino}hexanoate(1.3 g, 0.577 mmol) in a mixture of anhydrous DCM (5 mL) and anhydrousMeOH (15 mL) was added sodium methoxide (1.16 mL of a 0.5M soln in MeOH,0.577 mmol) and the resulting mixture stirred at room temperature for 3days. Mixture evaporated to a volume of ˜5 mL and added dropwise tostirring anhydrous acetonitrile (80 mL). Mixture centrifuged at 3500 rpmfor 30 mins, decanted the solvent and solid re-suspended in acetonitrile(80 mL). Mixture centrifuged at 3500 rpm for 30 mins, decanted thesolvent and solid air dried under a stream of dry nitrogen to give thetitle compound (650 mg, 100%). UPLC Method B: calculated for C₅₇H₈₃NO₂₂1133.54, observed m/e: 1134.64 [M+1]; Rt=2.08 min.

Step C: methyl6-{[3-(-α-L-fucopyranosyl)propyl](2-[2-{-α-D-mannopyranosyl-(1→3)-[-α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl}-oxy]ethyl)amino}hexanoatehydrochloride

To a solution of methyl6-{[3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)propyl](2-[2-{-α-D-mannopyranosyl-(1→3)-[-α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl}-oxy]ethyl)amino}hexanoate(650 mg, 0.577 mmol) in methanol (5 mL) was added conc. HCl (0.142 mL,1.73 mmol), flushed with nitrogen and 10% palladium on carbon (61 mg)added and stirred under a balloon of hydrogen for 3 hours. Filteredthrough a 0.4 micron syringe tip filter and the filtrate evaporated. Theresidue was dissolved in water (4 mL) and lyophilized to give the titlecompound (531 mg, 100%). UPLC Method B: calculated for C₃₉H₆₆N₂O₂₄863.40, observed m/e: 864.43 [M+1]; Rt=1.64 min.

Step D:3-(2-{([α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy)ethyl}{6-(2,5-dioxopyrrolidin-1-yl)-6-oxohexyl}amino)propylα-L-fucopyranoside

Prepared from methyl6-{[3-(-α-L-fucopyranosyl)propyl](2-[2-{-α-D-mannopyranosyl-(1→3)-[-α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]-oxy]ethyl)amino}hexanoatehydrochloride according to the procedures outlined for ML-XX steps F andG. UPLC Method B: calculated for C₃₉H₆₆N₂O₂₄ 946.40, observed m/e:947.51 [M+1]; Rt=3.55 min.

Example 58

The synthesis of oligosaccharide linker4-(2-{([α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy)ethyl}6-(2,5-dioxopyrrolidin-1-yl)-6-oxohexyl}amino)butylα-L-fucopyranoside (ML-58) having the following structure is described.

Step A: 3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)propyl methanesulfonate

To a solution of 3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)propanol [ML-36step D] (10.6 g, 22.2 mmol) and Hunig's Base (4.66 mL, 26.7 mmol) inanhydrous DCM (100 mL) cooled in an ice bath was added dropwisemethanesulfonyl chloride (1.9 mL, 24.5 mmol). After complete additionthe mixture was stirred at ice bath temperature for 1 hour. Mixturewashed with water (100 mL), sat. NaCl (50 mL); dried over Na₂SO₄,filtered and evaporated to give the title compound (12.6 g, 100%). ¹HNMR (CDCl₃) δ 7.37 (15H, m), 4.80 (2H, m), 4.68 (2H, m), 4.63 (1H, d,J=11.8 Hz), 4.53 (1H, J=11.8 Hz), 4.22 (2H, m), 4.00 (1H, d, J=9.8 Hz),3.94 (1H, m), 2.99 (3H, s), 1.88 (1H, m), 1.75 (2H, m), 1.62 (1H, m),1.31 (3H, d, J=6.6 Hz).

Step B: 3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)butanenitrile

To a solution of 3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)propylmethanesulfonate (3 g, 5.4 mmol) in anhydrous DMF (30 mL) was addedsodium azide (422 mg, 6.49 mmol) and the resulting mixture heated at 60°C. overnight. Mixture cooled and diluted with water (100 mL) andextracted with Et₂O (3×30 mL); combined Et₂O layers washed with sat.NaCl (30 mL); dried over Na₂SO₄, filtered and evaporated. The residuewas purified by silica gel column chromatography (Teledyne Isco; 120 g)eluent: gradient 0-70% EtOAc in Hexanes to give the title compound (6 g,76%). ¹H NMR (CDCl₃) δ 7.29 (15H, m), 4.80 (2H, m), 4.71 (1H, d, J=11.8Hz), 4.69 (1H, d, J=11.8 Hz), 4.65 (1H, d, J=11.8 Hz), 4.54 (1H, d,J=11.8 Hz), 3.95 (2H, m), 3.79 (3H, m), 2.37 (2H, m), 1.80 (2H, m), 1.62(2H, m), 1.31 (3H, d, J=6.6 Hz).

Step C: 3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)butanoic acid

A mixture of 3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)butanenitrile (6 g,12.36 mmol) in ethanol (100 mL) was treated with water (100 mL) and 5NNaOH (25 mL, 124 mmol) and the resulting mixture heated at reflux for 3days. Mixture cooled and ethanol removed by evaporation, the remainingaqueous was acidified by the addition of conc. HCl and extracted withEtOAc (3×100 mL); combined EtOAc layers washed with sat. NaCl (100 mL),dried over Na₂SO₄, filtered and evaporated to give the title compound(5.2 g, 83%) as a light yellow oil. ¹H NMR (CDCl₃) δ 7.40-7.30 (15H, m),4.79 (2H, m), 4.71 (1H, d, J=12.0 Hz), 4.65 (2H, m), 4.53 (1H, d, J=11.8Hz), 4.01 (1H, m), 3.92 (1H, m), 3.83 (1H, m), 3.78 (2H, m), 2.39 (2H,m), 1.75 (2H, m), 1.59 (2H, m), 1.30 (3H, d, J=6.6 Hz).

Step D: 4-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)butan-1-ol

To an ice bath cooled solution of3-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)butanoic acid (5.2 g, 10.3 mmol)in anhydrous THF (100 mL) was added slowly borane-tetrahydrofurancomplex (12.3 mL of a 1M soln in THF, 12.3 mmol) and the resultingmixture allowed to warm to room temperature and stirred for 3 days.Mixture quenched by the addition of methanol (5 mL) and diluted withsat. NaCl (200 mL) and extracted with EtOAc (2×150 mL); combined EtOAclayers dried over Na₂SO₄, filtered and evaporated. Residue purified bysilca gel column chromatography (Teledyne Isco: 120 g) eluent: gradient0-100% EtOAc in Hexanes to give the title compound (1.73 g, 34%) as aclear oil. ¹H NMR (CDCl₃) δ 7.40-7.28 (15H, m), 4.79 (2H, m), 4.71 (1H,d, J=12.1 Hz), 4.66 (2H, m), 4.54 (1H, d, J=11.9 Hz), 3.99 (1H, m), 3.93(1H, m), 3.80 (3H, m), 3.65 (2H, t, J=6.5 Hz), 1.67 (2H, m), 1.60-1.41(4H, m), 1.30 (3H, d, J=6.6 Hz).

Step E: 4-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)butanal

To a solution of 4-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)butan-1-ol(1.73 g, 3.53 mmol) in DCM (50 mL) was added Dess-Martin reagent (2.24g, 5.29 mmol) and the resulting mixture stirred at room temperature for3 hours. Mixture washed with sat. NaHCO₃ (100 mL); dried over Na₂SO₄,filtered and evaporated. The residue was purified by silica gel columnchromatography (Teledyne Isco: 80 g) eluent: gradient 0-80% EtOAc inHexanes to give the title compound (1.24 g, 72%). ¹H NMR (CDCl₃) δ 9.77(1H, s), 7.40-7.28 (15H, m), 4.80 (1H, d, J=12.0 Hz), 4.78 (1H, J=12.0Hz), 4.71 (1H, d, J=12.0 Hz), 4.66 (2H, m), 4.54 (1H, d, J=11.8 Hz),3.98 (1H, m), 3.92 (1H, m), 3.83-3.76 (3H, m), 2.43 (2H, m), 1.80-1.63(2H, m), 1.62-1.48 (2H, m), 1.30 (3H, d, J=6.6 Hz).

Step F:4-(2-{([α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl]oxy)ethyl}{6-(2,5-dioxopyrrolidin-1-yl)-6-oxohexyl}amino)butylα-L-fucopyranoside

Prepared from 4-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)butanal accordingto the procedures outlined for ML-57. UPLC Method B: calculated forC₄₀H₆₈N₂O₂₄ 960.42, observed m/e: 961.48 [M+1]; Rt=3.46 min.

Example 59

The synthesis of oligosaccharide linker2-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}[3-(α-L-fucopyranosyl)propyl]amino)ethylα-D-mannopyranoside (ML-59) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-36 substituting 2-aminoethyl2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside for 2-aminoethyl3,4,6-tri-O-acetyl-2-(acetylamino)-2-deoxy-β-D-glucopyranoside in StepF. UPLC Method B: calculated for C₂₇H₄₆N₂O₁₄ 622.29, observedm/e=623.3231 [M+1]; Rt=1.16 min.

Example 60

The synthesis of oligosaccharide linker2-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}[3-(α-L-fucopyranosyl)propyl]amino)ethylβ-D-mannopyranoside (ML-60) having the following structure is described.

Step A: benzyl (2-((4,6-di-O-benzoyl-β-Dgalactopyranosyl)oxy)ethyl)carbamate

To benzyl (2-((4,6-di-O-benzoyl-β-D galactopyranosyl)oxy)ethyl)carbamate(9.4 g, 26.3 mmol) was added 3 g of 4 Å powdered molecular sieves andmixture suspended in anhydrous toluene (100 mL). To this mixture wasadded dibutyltin (IV) oxide (14.21 g, 57.1 mmol) and the resultingmixture heated at 95° C. for 5 hours. The mixture was allowed to cool toroom temperature then cooled in an ice bath and benzoyl chloride (6.66mL, 57.3 mmol) added dropwise. Fine white precipitate formed, anhydrousacetonitrile (15 mL) added and stirred at room temperature for 48 hrs.Mixture evaporated and the residue was purified by column chromatographyon silica gel (Teledyne Isco: 330 g), eluent: 0 to 50% EtOAc in Hexanes(8cv); and 50% EtOAc in Hexanes (10cv) to give the title compound (11.56g, 78%) as a white solid. ¹H NMR (CDCl₃) δ 8.11 (2H, d, J=7.7 Hz), 8.04(2H, dd, J=7.9 and 1.4 Hz), 7.58 (2H, m), 7.45 (4H, m), 7.36-7.29 (5H,m), 5.53 (1H, m), 5.14 (1H, dd, J=10.1 and 3.3 Hz), 5.06 (2H, s), 4.64(1H, dd, J=11.5 and 6.3 Hz), 4.55 (1H, dd, J=11.5 and 6.6 Hz), 4.4 (1H,d, J=7.7 Hz), 4.24 (1H, t, J=4.2 Hz), 4.08 (1H, t, J=8.8 Hz), 3.93 (2H,m), 3.76 (1H, m), 3.48 (1H, m), 3.37 (1H, m), 3.28 (1H, d, J=3.3 Hz),2.80 (1H, d, J=5.3 Hz).

Step B: benzyl(2-((((3,5-bis(trifluoromethyl)sulfonyl)oxy)-4,6-di-O-benzoyl-fl-Dgalactopyranosyl)oxy)ethyl)carbamate

To a solution of benzyl (2-((4,6-di-O-benzoyl-β-Dgalactopyranosyl)oxy)ethyl)carbamate (11.56 g, 20.44 mmol) dissolved inDCM (200 mL) cooled to −15° C. was added pyridine (21.5 mL, 266 mmol)followed by slow addition of triflic anhydride (10.36 mL, 61.3 mmol) andthe resulting mixture allowed to warm to 0° C. over 3 hours. Mixturediluted with further DCM (200 mL) and washed with ice cold 1N HCl (500mL), ice cold sat. NaHCO₃ (500 mL) and ice cold sat. NaCl (500 mL);dried over Na₂SO₄, filtered and evaporated to give the title compound(16.9 g, 100%) as a yellow foam. ¹H NMR (CDCl₃) δ 8.16 (2H, dd, J=8.0and 1.4 Hz), 8.04 (2H, dd, J=8.1 and 1.4 Hz), 7.65 (2H, m),7.52 (2H, m),7.50 (2H, m), 7.39 (4H, m), 7.33 (1H, m), 5.55 (1H, dd, J=10.4 and 3.1Hz), 5.50 (1H, d, J=3.1 Hz), 5.35 (1H, t, J=6.4 Hz), 5.14 (2H, s), 5.12(1H, m), 4.77 (1H, d, J=7.9 Hz), 4.73 (1H, m), 4.28 (2H, m), 4.06 (1H,m), 3.80 (1H, m), 3.55 (1H, m), 3.47 (1H, m).

Step C: benzyl (2-((3,5-di-O-acetyl-4,6-di-O-benzoyl-β-Dmannopyranosyl)oxy)ethyl)carbamate

To a solution of benzyl(2-((((3,5-bis(trifluoromethyl)sulfonyl)oxy)-4,6-di-O-benzoyl-β-Dgalactopyranosyl)oxy)ethyl)carbamate (16.9 g, 20.37 mmol) in anhydroustoluene (100 mL), was added a solution of tetra butylammonium acetate(25 g, 82.9 mmol) in a mixture of toluene (150 mL) and DMF (4 mL) wasadded and the resulting mixture stirred at room temperature overnight.Diluted with of CH₂Cl₂ (30 mL) and washed with sat. NaCl (2×100 mL),dried over MgSO₄, filtered and evaporated. The residue was purified bysilica gel column chromatography (Teledyne Isco: 330 g) eluent: 0-50%EtOAc/Hexane (10cv) then 50% EtOAc/Hexane (5cv) to give the titlecompound (8 g, 60.5%). ¹H NMR (CDCl₃) δ 8.10 (2H, m), 7.98 (2H, dd,J=8.1 and 1.4 Hz), 7.61 (2H, m), 7.48 (2H, t, J=7.8 Hz), 7.46 (2H, t,J=7.7 Hz), 7.37 (4H, m), 7.33 (1H, m), 5.70 (1H, d, J=3.3 Hz), 5.61 (1H,t, J=10.0 Hz), 5.29 (1H, dd, J=10.0 and 3.3 Hz), 5.26 (1H, m), 5.10 (2H,s), 4.79 (1H, s), 4.64 (1H, dd, J=12.1 and 2.7 Hz), 4.47 (1H, dd, J=12.1and 5.8 Hz), 3.94 (2H, m), 3.74 (1H, m), 3.48 (1H, m), 3.37 (1H, m),2.15 (3H, s), 2.00 (3H, s).

Step D: 2-aminoethyl 3,5-di-O-acetyl-4,6-di-O-benzoyl-β-Dmannopyranoside

To a nitrogen flushed solution of benzyl(2-((3,5-di-O-acetyl-4,6-di-O-benzoyl-b-Dmannopyranosyl)oxy)ethyl)carbamate (8 g, 12.31 mmol) in EtOAc (100 ml)was added 10% palladium on carbon (1.31 g) and the resulting mixturestirred under a balloon of hydrogen overnight. The mixture was filteredthrough Celite and the filtrate evaporated to give the title compound(6.3 g, 99%) as a light yellow foam. ¹H NMR (CDCl₃) δ 8.11 (2H, m), 7.90(2H, m), 7.60 (2H, m), 7.47 (4H, m), 5.72 (1H, ddd, J=9.2, 3.2 and 1.1Hz), 5.59 (1H, t, J=9.7 Hz), 5.33 (1H, ddd, J=10.1, 4.8 and 3.3 Hz),4.85 (1H, dd, J 16.0 and 1.1 Hz), 4.65 (1H, m), 4.45 (1H, ddd, J=12.1,5.7 and 2.0 Hz), 3.95 (2H, m), 3.73 (1H, m), 3.09 (2H, bs), 2.90 (1H,m), 2.15 (3H, s), 1.99 (3H, s).

Step E:2-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}[3-(α-L-fucopyranosyl)propyl]amino)ethylα-D-mannopyranoside

The title compound was prepared using procedures analogous to thosedescribed for ML-36 substituting 2-aminoethyl3,5-di-O-acetyl-4,6-di-O-benzoyl-β-D mannopyranoside for 2-aminoethyl3,4,6-tri-O-acetyl-2-(acetylamino)-2-deoxy-β-D-glucopyranoside in StepF. UPLC Method B: calculated for C₂₇H₄₆N₂O₁₄ 622.29, observedm/e=623.3536 [M+1]; Rt=1.13 min.

Example 61

The synthesis of oligosaccharide linker2-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-2-[(α-L-fucopyranosyl)oxy]ethyl)-α-D-mannopyranoside(ML-61) having the following structure is described.

Step A:2-{[2-(2,3,4-tri-O-acetyl-α-L-fucopyranosyl)ethyl]amino}ethyl-2,3,4,6-tetra-O-acetyl-α-D-mannoopyranoside

To a mixture of 2-oxoethyl 2,3,4,6 tetra-O-acetyl-α-D-mannopyranoside(1.3 g, 3.33 mmol) and 2-aminoethyl2,3,4-tri-O-acetyl-α-L-fucopyranoside (2.22 g, 6.7 mmol) in anhydrousDCM (20 mL) was added TFA (0.257 mL, 3.3 mmol) and mixture stirred atroom temperature for 10 mins then sodium triacetoxyborohydride (1.41 g,6.66 mmol) added and mixture stirred at room temperature overnight.Mixture evaporated and the residue partitioned between EtOAc (50 mL) andsat. NaHCO₃ (100 mL); organic layer washed with sat. NaCl (50 mL); driedover Na₂SO₄; filtered and evaporated. The residue purified by reversephase silica gel column chromatography (Teledyne Isco: C18 275 g)eluent: gradient 10-100% CH₃CN in water to give the title compound (667mg, 28%). ¹H NMR (CDCl₃) δ 5.37 (1H, dd, J=10.0 and 3.5 Hz), 5.32 (1H,d, J=9.8 Hz), 5.29 (1H, dd, J=3.4 and 1.9 Hz), 5.26 (1H, dd, J=3.5 and1.1 Hz), 5.20 (1H, dd J=10.5 and 7.9 Hz), 5.04 (1H, dd, J 10.5 and 3.4Hz), 4.87 (1H, d, J=1.8 Hz), 4.50 (1H, d, J=7.9 Hz), 4.32 (1H, dd,J=12.3 and 2.5 Hz), 4.13 (1H, dd, J=12.2 and 2.5 Hz), 4.04 (1H, m), 4.00(1H, m), 3.86-3.79 (2H, m), 3.69 (1H, m), 3.59 (1H, m), 2.91-2.85 (4H,m), 2.19 (3H, s), 2.18 (3H, s), 2.13 (3H, s), 2.09 (3H, s), 2.07 (3H,s), 2.02 (3H, s), 2.01 (3H, s), 1.25 (3H, d, J=6.4 Hz).

Step B: benzyl6-{[2-(2,3,4-tri-O-acetyl-α-L-fucopyranosyl)ethyl](2-{[2,3,4,6-tetra-O-acetyl-α-D-mannoopyran]oxy}ethyl)amino}hexanoate

To a solution of2-{[2-(2,3,4-tri-O-acetyl-α-L-fucopyranosyl)ethyl]amino}ethyl-2,3,4,6-tetra-O-acetyl-α-D-mannoopyranoside(667 mg, 0.943 mmol) and benzyl 6-oxohexanoate (311 mg, 1.41 mmol) inDCM (6 mL) was added acetic acid (0.054 mL, 0.943 mmol) mixture stirredat room temperature for 10 mins then sodium triacetoxyborohydride (400mg, 1.89 mmol) added and mixture strirred at room temperature overnight.UPLC-MS shows complete conversion. Mixture evaporated and the residuepartitioned between EtOAc (30 mL) and sat. NaHCO₃ (40 mL); organic layerwashed with sat. NaCl (20 mL); dried over Na₂SO₄, filtered andevaporated. The residue was purified by reverse phase silica gel columnchromatography (Teledyne Isco: C18 40 g) eluent: gradient 5-100% CH₃CNin water to give the title compound (434 mg, 50%). ¹H NMR (CDCl₃) δ 7.37(5H, m), 5.35 (1H, dd, J=9.9 and 3.1 Hz), 5.32 (1H, d, J=9.2 Hz), 5.25(2H, dd, J=3.2 and 1.7 Hz), 5.19 (1H, dd, J=10.5 and 7.9 Hz), 5.14 (2H,s), 5.04 (1H, dd, J=10.4 and 3.5 Hz), 4.85 (1H, d, J=1.7 Hz), 4.51 (1H,d, J=7.9 Hz), 4.32 (1H, dd, J=12.2 and 5.1 Hz), 4.12 (1H, dd, J=12.2 and2.5 Hz), 4.04 (1H, m), 3.93 (1H, dt, J=9.9 and 5.9 Hz), 3.85 (1H, m),3.72 (1H, dt, J=10.1 and 6.0 Hz), 3.59 (1H, dt, J=9.9 and 6.6 Hz), 3.51(1H, m), 2.74-2.71 (4H, m), 2.50 (2H, t, J=7.4 Hz), 2.39 (2H, t, J=7.5Hz), 2.19 (3H, s), 2.18 (3H, s), 2.13 (3H, s), 2.07 (3H, s), 2.06 (3H,s), 2.01 (3H, s), 2.00 (3H, s), 1.70-1.66 (4H, m), 1.44 (2H, m), 1.32(2H, m), 1.24 (3H, d, J=6.4 Hz).

Step C:2-({6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-2-[(α-L-fucopyranosyl)oxy]ethyl)-α-D-mannopyranoside

The title compound was prepared using procedures analogous to thosedescribed for ML-35 in step B substituting benzyl6-{[2-(2,3,4-tri-O-acetyl-α-L-fucopyranosyl)ethyl](2-{[-2,3,4,6-tetra-O-acetyl-α-D-mannoopyran]oxy}ethyl)amino}hexanoatefor benzyl 6-(bis{2-[(2,3,4-tri-O-benzoyl-α-L-fucopyranosyl)oxy]ethyl}amino)hexanoate.UPLC Method B: calculated for C₂₆H₄₄N₂O₁₅ 624.27, observed m/e=625.2990[M+1]; Rt=1.12.

Example 62

The synthesis of oligosaccharide linkerN,N′-Bis{2-[(α-L-fucopyranosyl)oxy]ethyl}-1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexanoyl}pyrrolidine-(2R,5R)-2,5-dicarboxamide(ML-62) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-6 substituting (2R,5R)-pyrrolidine-2,5-dicarboxylicacid for 2,2′-{[6-(benzyloxy)-6-oxohexanoyl]imino}diacetic acid in StepC. UPLC Method B: m/e=736.36 [M+1]; Rt=2.22 min.

Example 63

The synthesis of oligosaccharide linker N,N-Bis{2-[(α-L-fucopyranosyl)oxy]ethyl}-1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexanoyl}(piperidine-4,4-diyl)diacetamide(ML-63) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-6 substituting 2,2′-(piperidine-4,4-diyl)diacetic acidfor 2,2′-{[6-(benzyloxy)-6-oxohexanoyl]imino}diacetic acid in Step C.UPLC Method B: m/e=805.38 [M+1]; Rt=2.35 min.

Example 64

The synthesis of oligosaccharide linker1-{6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexanoyl}-N,N′-bis{2-[(α-L-fucopyranosyl)oxy]ethyl}piperidine-cis-3,4-dicarboxamide(ML-64) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-17 substituting 3,4-pyridinedicarboxylic acid for3,5-pyridinedicarboxylic acid as the starting material in step A. UPLCMethod F: m/e=777.3660 [M+1]; Rt=2.15 min.

Example 65

The synthesis of oligosaccharide linker 2,5-dioxopyrrolidin-1-yl6-((3R,4R)-3,4-bis((2-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)ethyl)carbamoyl)piperidin-1-yl)hexanoate(ML-65) having the following structure is described.

Step A:(3R,4R)—N3,N4-bis(2-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)ethyl)piperidine-3,4-dicarboxamide

The title compound was prepared using the procedure analogous to thatdescribed for ML-17 Steps A and B, substituting 3,4-pyridinedicarboxylicacid for 3,5-pyridinedicarboxylic acid as the starting material in stepA. UPLC Method F: m/e=552.2733 [M+1]; Rt=1.37 min.

Step B: benzyl6-((3R,4R)-3,4-bis((2-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)ethyl)carbamoyl)piperidin-1-yl)hexanoate

(3R,4R)—N3,N4-bis(2-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)ethyl)piperidine-3,4-dicarboxamide(118 mg, 0.214 mmol) was dissolved in THF (2 mL), to which benzyl6-oxohexanoate (70.7 mg, 0.321 mmol) in THF (0.5 mL) was added, followedby sodium triacetoxyborohydride (136 mg, 0.642 mmol) and acetic acid(3.67 μL, 0.064 mmol) were added, and the mixture was stirred at roomtemperature for 3 h. The product was isolated by preparativereverse-phase chromatography on C-18 column, using a gradient of 0-30%of AcN in water. UPLC Method F: m/e=756.4241 [M+1]; Rt=3.05 min

Step C: The synthesis of oligosaccharide linker 2,5-dioxopyrrolidin-1-yl6-((3R,4R)-3,4-bis((2-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)ethyl)carbamoyl)piperidin-1-yl)hexanoate

The title compound was prepared using procedure analogous to thosedescribed for ML-1 Steps C and D substituting benzyl6-((3R,4R)-3,4-bis((2-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)ethyl)carbamoyl)piperidin-1-yl)hexanoatefor benzyl6-({2-[(α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}amino)-6-oxohexanoatein Step C and substituting 6-((3R,4R)-3,4-bis((2-(((2R,3 S,4R,5S,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)ethyl)carbamoyl)piperidin-1-yl)hexanoicacid for6-({2-[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)oxy]ethyl}amino)-6-oxohexanoicacid in Step D: UPLC Method F: m/e=763.7796 [M+1]; Rt=2.15 min.

Example 66

The synthesis of oligosaccharide linker 2,5-dioxopyrrolidin-1-yl6-((2R,5R)-2,5-bis((2-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)ethyl)carbamoyl)piperidin-1-yl)-6-oxohexanoate(ML-66) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-17 substituting 2,5-pyridinedicarboxylic acid for3,5-pyridinedicarboxylic acid as the starting material in step A. UPLCMethod A: UPLC m/e=777.0 [M+1]; Rt=0.5 min.

Example 67

The synthesis of oligosaccharide linker 2,5-dioxopyrrolidin-1-yl6-(((R)-1,4-dioxo-1-((2-(((2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)amino)-4-((2-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)ethyl)amino)butan-2-yl)amino)-6-oxohexanoate(ML-67) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-20 substituting Z-Glu-γ-Bn for Z-ASP(OBZL)-OH andsubstituting 2-aminoethylα-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosidefor 2-aminoethyl α-D-mannopyranoside in Step A. UPLC Method B:m/e=753.26 [M+1]; Rt=1.59 min.

Example 68

The synthesis of oligosaccharide linker (ML-68) having the followingstructure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-20 substituting Z-Glu-γ-Bn for Z-ASP(OBZL)-OH in StepA. UPLC Method B: m/e=1077.52 [M+1]; Rt=2.93 min.

Example 69

The synthesis of oligosaccharide linker 2,5-dioxopyrrolidin-1-yl6-(2-(bis(-[(α-L-fucopyranosyl)oxy)ethyl)amino)acetamido)hexanoate(ML-69) having the following structure is described.

Step A: 1,2,3,4-tetrakis(oxy)tetrakis(trimethylsilane) L-Fucose

To a solution of L-Fucose (4.0 g, 24.37 mmol, 1.0 eq) in DMF (25 mL) at0° C. was added TEA (17.32 mL, 124 mmol, 5.1 eq). To above mixture wasadded TMS-Cl (15.88 mL, 125 mmol, 5.1 eq) dropwise. The reaction wasthen warmed to r.t. and stirred at r.t. for 4 hr. The reaction mixturewas poured to ice and hexance mixture (100 mL, 1:1). The mixture wasextracted with hexane (100 mL×3). The organic was washed with water (10ml×3), dried over MgSO4, filtered. The filtrated was concentrated anddried over high vacuum pump to give the titled compound as colorless oil(8.7 g, 19.2 mmol, 79%). 13C NMR (CDCl3, 125 MHz) δ 94.5 (1C), 70.6(1C), 69.6 (1C), 66.6 (1C), 39.6 (4C), 16.7 (Me), 0.67 (3Me), 0.43(3Me), 0.29 (3Me), 0.16 (3Me); ¹H NMR (CDCl3, 500 MHz) δ 5.0 (s, 1H),4.0 (m, 1H), 3.8 (1H), 3.6 (1H), 1.0 (d, 3H), 0-0.2 (m, 36H).

Step B: benzyl 6-(2-(bis(2-hydroxyethyl)amino)acetamido)hexanoate

To a solution of 2-(bis(2-hydroxyethyl)amino)acetic acid (500 mg, 3.06mmol, 1.0 eq) in DMF (10 mL) at zero degree, was added TSTU (1107 mg,3.68 mmol, 1.2 eq) followed by TEA (0.512 mL, 3.68 mmol, 1.2 eq). Thereaction was warmed to rt and stirred at that temperature for 2 h. Toabove mixture was added L-000503048-001W001 (1447 mg, 3.68 mmol, 1.2 eq)pre-mixed with TEA (0.512 ml, 3.66 mmol). The reaction was stirred at rtfor 18 hr. UPLC indicated formation of desired product. DMF was removedunder reduced pressure. The crude was purified by C18 Reverse phasechromatography (eluted with 0-30% ACN/water in 16 CV). Fractionscontaining desired product were combined, concentrated and lyo to givethe titled compound as colorless syrup. UPLC Method B: m/e=367.2356[M+1]; Rt=3.54 min.

Step C: benzyl6-(2-(bis(2-[(α-L-fucopyranosyl)oxy)ethyl)amino)acetamido)hexanoate

To a solution of benzyl6-(2-(bis(2-hydroxyethyl)amino)acetamido)hexanoate (210 mg, 0.573 mmo,1.0 eq) in DCM (10 mL) at zero degree, was added TBAI (1820 mg, 4.93mmol, 8.6 eq), DIPEA (0.500 ml, 2.87 mmol, 5.0 eq). The mixture waswarmed to rt and stirred at rt for 30 min. To above solution was added1,2,3,4-tetrakis(oxy)tetrakis(trimethylsilane) L-Fucose (1557 mg, 3.44mmol, 6.0 eq) with iodotrimethylsilane (0.390 ml, 2.87 mmol, 5.0 eq) inDCM (10 ml) dropwise. The mixture was stirred at rt for 18 hr. UPLCindicated formation of desired product. Remove DCM and added MeOH (10ml) and Dowex H+ resin till pH ˜2. Stirred at rt for 1 h. filteredthrough a pad of celite. The filtrated was concentrated and purified byC18 Reverse phase chromatography (eluted with 0-30% ACN/water in 16 CV).Fractions containing desired product were combined, concentrated and lyoto give the titled compound as colorless syrup (40 mg, 0.061 mmol,10.6%). UPLC Method B: m/e=659.3745 [M+1]; Rt=3.36 min.

Step D:6-(2-(bis(2-[(α-L-fucopyranosyl)oxy)ethyl)amino)acetamido)hexanoic acid

To a solution of benzyl6-(2-(bis(2-[(α-L-fucopyranosyl)oxy)ethyl)amino)acetamido)hexanoate (40mg, 0.061 mmol) in water (5 ml), was added Pd/C (30.5 mg, 0.029 mmol).The reaction was stirred under H2 balloon for 18 hr. UPLC indicatedformation of desired product. The above solution was diluted with MeOH(5 mL), filtered through a pad of celite, concentrated and lyo to givethe titled compound as colorless syrup (20 mg, 6.1%). UPLC Method B:m/e=569.3191 [M+1]; Rt=1.93 min.

Step E: 2,5-dioxopyrrolidin-1-yl6-(2-(bis(-[(α-L-fucopyranosyl)oxy)ethyl)amino)acetamido)hexanoate

To a solution of6-(2-(bis(2-[(α-L-fucopyranosyl)oxy)ethyl)amino)acetamido)hexanoic acid(20 mg, 0.037 mmol, 1.0 eq) in DMF (1 mL) was added TSTU (16.68 mg,0.055 mmol, 1.5 eq) followed by Hunig'sBase (7.74 μl , 0.044 mmol, 1.2eq). The reaction was stirred at rt for 1 h. TLC (4/1/1/1EtOAc/MeOH/ACN/water) indicated no starting material left. UPLCindicated formation of pdt. Remove DMF under reduced pressure. The crudeproduct was used without purification. UPLC Method B: m/e=666.3351[M+1]; Rt=2.28 min.

Example 70

The synthesis of oligosaccharide linker 2,5-dioxopyrrolidin-1-yl6-(2-(bis(-[(α-L-fucopyranosyl)oxy)propyl)amino)acetamido)hexanoate(ML-70) having the following structure is described.

Step A: benzyl 6-(bis(3-hydroxypropyl)amino)-6-oxohexanoate

To a solution of 3,3′-azanediylbis(propan-1-ol) (1000 mg, 7.51 mmol, 1.0eq) was in DMF (10 ml), was added benzyl (2,5-dioxopyrrolidin-1-yl)adipate (2503 mg, 7.51 mmol, 1.0 eq) followed by TEA (1.046 ml, 7.51mmol, 1.0 eq). The reaction was stirred at 25 deg for 18 hr. UPLCindicated formation of desired product. DMF was removed under reducedpressure. The crude was purified by C18 Reverse phase chromatograph(eluted with 0-40% ACN/water in 16 CV). Fractions containing desiredproduct were combined and concentrated to give the titled compound ascolorless oil (1.55 g, 4.41 mmol, 58.7%). UPLC Method B: m/e=352.2171[M+1]; Rt=3.47 min. ¹H NMR (CDCl3, 500 MHz) δ 7.3-7.5 (m, 5H), 5.15 (m,2H), 3.65 (m, 2H), 3.40 (m, 5H), 2.66 (s, 3H), 2.45 (m, 3H), 2.29 (s,1H), 1.84 (s, 1H), 1.70 (m, 7H).

Step B: benzyl6-(2-(bis(2-[(α-L-fucopyranosyl)oxy)propyl)amino)acetamido)hexanoate

The title compound was prepared using procedures analogous to thosedescribed for ML-69 substituting benzyl6-(bis(3-hydroxypropyl)amino)-6-oxohexanoate for benzyl6-(2-(bis(2-hydroxyethyl)amino)acetamido)hexanoate in Step C. UPLCMethod B: m/e=644.3454 [M+1]; Rt=3.28 min.

Step C:6-(2-(bis(2-[(α-L-fucopyranosyl)oxy)propyl)amino)acetamido)hexanoic acid

The title compound was prepared using procedures analogous to thosedescribed for ML-69 substituting benzyl6-(2-(bis(2-[(α-L-fucopyranosyl)oxy)propyl)amino)acetamido)hexanoate forbenzyl6-(2-(bis(2-[(α-L-fucopyranosyl)oxy)ethyl)amino)acetamido)hexanoate inStep D. UPLC Method B: m/e=554.3076 [M+1]; Rt=2.13 min.

Step E: 2,5-dioxopyrrolidin-1-yl6-(2-(bis(-[(α-L-fucopyranosyl)oxy)propyl)amino)acetamido)hexanoate

The title compound was prepared using procedures analogous to thosedescribed for ML-69 substituting6-(2-(bis(2-[(α-L-fucopyranosyl)oxy)propyl)amino)acetamido)hexanoic acidfor 6-(2-(bis(2-[(α-L-fucopyranosyl)oxy)ethyl)amino)acetamido)hexanoicacid in Step E. UPLC Method B: m/e=651.3166 [M+1]; Rt=2.41 min.

Example 71

The synthesis of oligosaccharide linker benzyl6-(2-(bis(2-[(α-L-fucopyranosyl)oxy)butyl)amino)acetamido)hexanoate(ML-71) having the following structure is described.

Step A: 4-(benzyoxy)-N-(4-(benzyloxy)butyl)butanamide

To a solution 4-(benzyloxy)butanoic acid (1 g, 5.15 mmol, 1.0 eq) in DMF(5 ml) at zero deg, was added TSTU (1.627 g, 5.41 mmol, 1.05 eq)followed by TEA (0.718 ml, 5.15 mmol, 1.0 eq). The reaction was warmedto rt and stirred at rt for 2 hr. To above reaction was added4-(benzyloxy)butan-1-amine (0.969 g, 5.41 mmol, 1.05 eq) followed by TEA(0.718 ml, 5.15 mmol, 1.0 eq). The reaction was stirred at rt for 18 hr.LC-MS showed formation of desired product. DMF was removed under reducedpressure. The crude was purified by silica gel column (120 g, elutedwith 0-15% MeOH/DCM in 16 CV). Fractions containing desired product werecombined and concentrated to give the titled compound (1.65 g, 4.64mmol, 90% yield). LC-MS Method A: m/e=356.70 [M+1]; Rt=1.22 min. ¹H NMR(CDCl3, 500 MHz) δ 7.2-7.4 (m, 10H), 5.98 (s, 1H), 4.51 (m, 4H), 3.53(m, 4H), 3.24 (m, 2H), 2.28 (m, 2H), 1.96 (m, 2H), 1.5-1.7 (m, 4H).

Step B: bis(4-(benzyloxy)butyl)amine

In a 200 mL round bottom flask, to a solution of4-(benzyoxy)-N-(4-(benzyloxy)butyl)butanamide (1.65 g, 4.64 mmol) in THF(5 ml) at zero deg, was added BH3.THF (13.93 ml, 13.93 mmol) dropwise.The reaction was warmed to rt and stirred at rt for 18 hr. TLC showedformation of pdt and disappear of starting material. The reaction wasquenched with aqueous saturated NH4Cl. The mixture was concentrated,dilute with EtOAc, shake with 1 N HCl, also wash with bicarbonate, brineand water. The organic layer was dried over MgSO4, filtered andconcentrated. The crude was used to next step without purification.LC-MS Method A: m/e=341.00 [M+1]; Rt=1.06 min.

Step C: 4,4′-azanediylbis(butan-1-ol)

To a solution of bis(4-(benzyloxy)butyl)amine (300 mg, 0.879 mmol) wasin a mixed solvent of Dioxane (5 ml)/Water (5 mL), was added PdOH2 (30.8mg, 0.044 mmol). The reaction was stirred under H2 at 40 PSI for 18 h.LC-MS showed no starting material and formation of desired product. Themixture was filtered through a pad of celite, washed with dioxane/water(10 mL, 1/1). The filtrate was concentrated and dried over high vacuumpump to give the titled compound (130 mg, 0.806 mmol, 92% yield). LC-MSMethod A: m/e=162.01 [M+1]; Rt=0.18 min.

Step D: benzyl6-(2-(bis(2-[(α-L-fucopyranosyl)oxy)butyl)amino)acetamido)hexanoate

The title compound was prepared using procedures analogous to thosedescribed for ML-69 substituting benzyl6-(bis(3-hydroxybutyl)amino)-6-oxohexanoate for benzyl6-(2-(bis(2-hydroxyethyl)amino)acetamido)hexanoate in Step C. UPLCMethod B: m/e=644.3454 [M+1]; Rt=3.28 min.

Step E:6-(2-(bis(2-[(α-L-fucopyranosyl)oxy)propyl)amino)acetamido)hexanoic acid

The title compound was prepared using procedures analogous to thosedescribed for ML-69 substituting benzyl6-(2-(bis(2-[(α-L-fucopyranosyl)oxy)butyl)amino)acetamido)hexanoate forbenzyl6-(2-(bis(2-[(α-L-fucopyranosyl)oxy)ethyl)amino)acetamido)hexanoate inStep D. UPLC Method B: m/e=554.3076 [M+1]; Rt=2.13 min.

Step F: 2,5-dioxopyrrolidin-1-yl6-(2-(bis(-[(α-L-fucopyranosyl)oxy)butyl)amino)acetamido)hexanoate

The title compound was prepared using procedures analogous to thosedescribed for ML-69 substituting6-(2-(bis(2-Rα-L-fucopyranosyl)oxy)butyl)amino)acetamido)hexanoic acidfor 6-(2-(bis(2-Rα-L-fucopyranosyl)oxy)ethyl)amino)acetamido)hexanoicacid in Step E. UPLC Method B: m/e=651.3166 [M+1]; Rt=2.41 min.

Step G: benzyl6-(2-(bis(2-[(α-L-fucopyranosyl)oxy)butyl)amino)acetamido)hexanoate

The title compound was prepared using procedures analogous to thosedescribed for ML-69 substituting benzyl6-(bis(3-hydroxybutyl)amino)-6-oxohexanoate for benzyl6-(2-(bis(2-hydroxyethyl)amino)acetamido)hexanoate in Step C. UPLCMethod B: m/e=644.3454 [M+1]; Rt=3.28 min.

Example 72

The synthesis of oligosaccharide linker 2,5-dioxopyrrolidin-1-yl6-(2,3-bis-2[2-(α-L-fucopyranosyl)oxyethyl)carbamoyl)cyclopropanecarboxylic)amino)acetamido))hexanoate(ML-72) having the following structure is described.

Step A:2,3-bis-2[2-(α-L-fucopyranosyl)oxyethyl)carbamoyl)cyclopropanecarboxylicacid

To a solution L-000719504-000×003 (353 mg, 2.027 mmol) in DMF (10 ml),was added EDC (816 mg, 4.26 mmol) and HOBT (93 mg, 0.608 mmol). Themixture was stirred at 25 deg for 30 min. To above mixture was added AEF(882 mg, 4.26 mmol). The mixture was stirred at 25 for 18 hr. UPLCindicated formation of desired product. DMF was removed under reducedpressure. The crude was purified by C18 reverse phase chromatograph(eluted with 0-30% ACN/water with 0.05% TFA in 37 min) Fractionscontaining desired product were combined and lyo to give the titledcompound (80 mg, 0.145 mmol, 7.14% yield). UPLC Method B: m/e=553.2539[M+1]; Rt=2.63 min.

Step B: benzyl6-(2,3-bis-2[2-(α-L-fucopyranosyl)oxyethyl)carbamoyl)cyclopropanecarboxylic)amino)acetamido) hexanoate

The title compound was prepared using procedures analogous to thosedescribed for ML-69 substituting2,3-bis-2[2-(α-L-fucopyranosyl)oxyethyl)carbamoyl)cyclopropanecarboxylicacid for 2-(bis(2-hydroxyethyl)amino)acetic acid in Step B. UPLC MethodB: m/e=756.3689 [M+1]; Rt=3.15 min.

Step C:6-(2,3-bis-2[2-(α-L-fucopyranosyl)oxyethyl)carbamoyl)cyclopropanecarboxylic)amino)acetamido)hexanoicacid

The title compound was prepared using procedures analogous to thosedescribed for ML-69 substituting for benzyl6-(2-(bis(2-Rα-L-fucopyranosyl)oxy)ethyl)amino)acetamido)hexanoate forbenzyl6-(2-(bis(2-[(α-L-fucopyranosyl)oxy)ethyl)amino)acetamido)hexanoate inStep D. UPLC Method B: m/e=666.3151 [M+1]; Rt=1.23 min.

Step D: 2,5-dioxopyrrolidin-1-yl6-(2,3-bis-2[2-(α-L-fucopyranosyl)oxyethyl)carbamoyl)cyclopropanecarboxylic)amino)acetamido))hexanoate

The title compound was prepared using procedures analogous to thosedescribed for ML-69 substituting6-(2,3-bis-2[2-(α-L-fucopyranosyl)oxyethyl)carbamoyl)cyclopropanecarboxylic)amino)acetamido)hexanoicacid for6-(2-(bis(2-[(α-L-fucopyranosyl)oxy)ethyl)amino)acetamido)hexanoic acidin Step E. UPLC Method B: m/e=763.3411 [M+1]; Rt=1.99 min.

Example 73

The synthesis of oligosaccharide linker 2,5-dioxopyrrolidin-1-yl6-oxo-(6-((3-alpha-D-mannopyranosyl)propyl-α-L-fucopyranosyl)ethyl]amino)hexanoate(ML-73) having the following structure is described.

Step A: per-TMS D-Mannose

The titled compound was prepared using procedures analogous to thosedescribed for ML-69 substituting D-Mannose for L-Fucose in Step A. ¹HNMR (CDCl3, 500 MHz) δ 4.9 (s, 1H), 3.5-3.9 (m, 6H), 0-0.3 (m, 45H).

Step B: 2,3,4,6-tetra-O-trimethylsilane D-mannopyranosyl

To the solution of per-TMS D-Mannose (5.4 g, 9.98 mmol) in DCM (25 ml)at zero degrees, was added iodotrimethylsilane (1.426 ml, 10.48 mmol).The reaction was warmed to rt and stirred at rt for 1 hr. Remove DCM byreduced pressure. The intermediate was used next step withoutpurification.

Step C: 3-Iodopropoxyl alpha-D-Mannopyranoside and 3-Iodopropoxylbeta-D-Mannopyranoside

To the solution of 2,3,4,6-tetrakis(trimethylsilane) D-mannopyranosyl(2.89 g, 4.99 mmol) in DCM (10 ml) at zero deg, was added oxetane (0.488ml, 7.49 mmol). The reaction was warmed to rt and stirred at rt for 5hr. DCM was removed by rotavap. The mixture was dissolved in MeOH (10mL). To above solution was added Dowex H+ resin till pH ˜2. The mixturewas stirred at rt for 1 hr. LC-MS indicated formation of desiredproduct. The mixture was filtered through a pad of celite, concentratedand purified by C8 reverse phase chromatography (eluted with 5-25%ACN/water with 0.05% TFA in 25 min) Fractions containing desired productwere collected and lyo to give 3-iodopropoxyl alpha-D-mannopyranoside(710 mg, 2.04 mmol, 40.8%) and 3-iodopropoxyl beta-D-mannopyranoside(420 mg, 1.21 mmol, 24.2%). LC-MS Method A: m/e=696.96 [M+1]; Rt=0.46min and Rt=0.53 min. ¹H NMR (CD3OD, 500 MHz) 3-iodopropoxylbeta-D-mannopyranoside: δ 4.54 (d, J=0.95 Hz, 1H), 3.95 (m, 1H),3.88-3.92 (m, 2H), 3.75 (m, 1H), 3.65 (m, 1H), 3.50 (m, 1H), 3.45 (m,1H), 3.3-3.4 (m, 2H), 3.23 (m, 1H), 2.1 (m, 2H). ¹H NMR (CD3OD, 500 MHz)3-iodopropoxyl alpha-D-mannopyranoside: δ 4.81 (m, 1H), 3.8-3.9 (m, 3H),3.6-3.8 (m, 3H), 3.5-3.6 (m, 2H), 3.3-3.4 (m, 2H), 2.1(m, 2H).

Step D: α-L-fucopyranosyl)ethyl]amino}propyl alpha-D-Mannopyranoside

To the solution of 3-iodopropoxyl alpha-D-mannopyranoside (220 mg, 0.632mmol) in DMF (5 mL), was added AEF (131 mg, 0.632 mmol) and LiOH (15.13mg, 0.632 mmol). The mixture was stirred at rt for 24 hr. UPLC indicatedformation of pdt. DMF was removed under reduced pressure. The crude wascarried to next step without purification. UPLC Method B: m/e=428.2252[M+1]; Rt=1.02 min.

Step E: benzyl6-oxo-(6-((3-alpha-D-mannopyranosyl)propyl-α-L-fucopyranosyl)ethyl]amino)hexanoate

The titled compound was prepared using procedures analogous to thosedescribed for ML-69 substituting α-L-fucopyranosyl)ethyl]amino}propylalpha-D-Mannopyranoside for α-L-fucopyranosyl)ethyl]amino}propylalpha-D-Mannopyranoside in Step A. UPLC Method B: m/e=646.3233 [M+1];Rt=3.13 min.

Step F:6-oxo-(6-((3-alpha-D-mannopyranosyl)propyl-α-L-fucopyranosyl)ethyl]amino)hexanoic acid

The titled compound was prepared using procedures analogous to thosedescribed for ML-69 substituting benzyl6-oxo-(6-((3-alpha-D-mannopyranosyl)propyl-α-L-fucopyranosyl)ethyl]amino)hexanoatefor benzyl6-(2-(bis(2-Rα-L-fucopyranosyl)oxy)ethyl)amino)acetamido)hexanoate inStep D. UPLC Method B: m/e=556.2731 [M+1]; Rt=1.77 min.

Step G: 2,5-dioxopyrrolidin-1-yl6-oxo-(6-((3-alpha-D-mannopyranosyl)propyl-α-L-fucopyranosyl)ethyl]amino)hexanoate

The title compound was prepared using procedures analogous to thosedescribed for ML-69 substituting6-oxo-(6-((3-alpha-D-mannopyranosyl)propyl-α-L-fucopyranosyl)ethyl]amino)hexanoic acid for6-(2-(bis(2-Rα-L-fucopyranosyl)oxy)ethyl)amino)acetamido)hexanoic acidin Step E. UPLC Method B: m/e=653.3008 [M+1]; Rt=2.09 min.

Example 74

The synthesis of oligosaccharide linker 2,5-dioxopyrrolidin-1-yl6-oxo-(6-((3-beta-D-mannopyranosyl)propyl-α-L-fucopyranosyl)ethyl]amino)hexanoate(ML-74) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-73 substituting beta-D-mannopyranose foralpha-D-mannose in Step A-F. UPLC Method B: m/e=653.3167 [M+1]; Rt=2.07min.

Example 75

The synthesis of oligosaccharide linker 2,5-dioxopyrrolidin-1-yl8-oxo-(6-((3-beta-D-mannopyranosyl)propyl-α-L-fucopyranosyl)ethyl]amino)octanediate(ML-75) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-73 substituting beta-D-mannopyranose foralpha-D-mannose in Step A-F and substituting benzyl8-(2,5-dioxopyrrolidin-1-yl) octanediate for benzyl(2,5-dioxopyrrolidin-1-yl) adipate. UPLC Method B: m/e=681.3568 [M+1];Rt=2.44 min.

Example 76

The synthesis of oligosaccharide linker 2,5-dioxopyrrolidin-1-yl8-oxo-(6-((3-alpha-D-mannopyranosyl)propyl-α-L-fucopyranosyl)ethyl]amino)octanediate(ML-76) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-73 substituting benzyl 8-(2,5-dioxopyrrolidin-1-yl)octanediate for benzyl (2,5-dioxopyrrolidin-1-yl) adipate. UPLC MethodB: m/e=681.3456 [M+1]; Rt=2.21 min.

Example 77

The synthesis of oligosaccharide linker 2,5-dioxopyrrolidin-1-yl9-oxo-(6-((3-alpha-D-mannopyranosyl)propyl-α-L-fucopyranosyl)ethyl]amino)nonanedioate(ML-77) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-73 substituting benzyl 9-(2,5-dioxopyrrolidin-1-yl)nonanedioate for benzyl (2,5-dioxopyrrolidin-1-yl) adipate. UPLC MethodB: m/e=695.3532 [M+1]; Rt=2.55 min.

Example 78

The synthesis of oligosaccharide linker 2,5-dioxopyrrolidin-1-yl10-oxo-(6-((3-alpha-D-mannopyranosyl)propyl-α-L-fucopyranosyl)ethyl]amino)decanedioate (ML-78) having the following structure is described.

The title compound was prepared using procedures analogous to thosedescribed for ML-73 substituting benzyl 10-(2,5-dioxopyrrolidin-1-yl)decanedioate for benzyl (2,5-dioxopyrrolidin-1-yl) adipate. UPLC MethodB: m/e=709.3766 [M+1]; Rt=2.79 min.

Example 79

The synthesis of oligosaccharide linker6-[(2,5-dioxopyrrolidin-1-yl)oxy]-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-β-D-mannopyranosyl]oxy}propyl)-6-oxohexanamide(ML-79) having the following structure is described.

Step A: 3-azidopropoxyl β-D-mannopyranoside

To the solution of 3-iodopropoxyl 3-D-mannopyranoside (2.0 g, 5.74 mmol)in DMF (10 ml), was added sodium azide (448 mg, 0.448 mmol). Thereaction was warmed up to 60 degrees and stirred at this temperature for12 hr under N2. LC-MS indicated formation of desired product. DMF wasremoved under reduced pressure. The crude was purified by C18 reversephase chromatography (eluted with 0-20% ACN/water in 16 CV, then 100%ACN in 2 CV, 0% ACN 2CV). Fractions containing desired pdt were combinedand lyo to give the titled compound (1.27 g, 4.82 mmol, 84% yield).LC-MS Method A: m/e=264.16 [M+1]; Rt=0.21. ¹H NMR (CD3OD, 500 MHz): δ4.53 (m, 1H), 4.02 (m, 1H), 3.97 (m, 2H), 3.65 (m, 2H), 3.56 (m, 1H),3.48 (m, 3H), 3.22 (m, 1H), 1.92 (m, 2H).

Step B: 2.4-benzoyl 3-azidopropoxyl β-D-mannopyranoside

To the solution 3-azidopropoxyl β-D-mannopyranoside (1030 mg, 3.91 mmol)in acetonitrile (15 ml) was added triethyl orthobenzoate (2.352 ml,10.17 mmol) followed by TFA (0.030 ml, 0.391 mmol) and in ACN (0.5 mL).The mixture was allowed to stir at room temperature for 1 hour. Rotavapto remove ACN. TFA (10% in water) (4.28 ml, 5.55 mmol) was added. Themixture was stirred at rt for 2 hours. The residue was purified bycolumn chromatography on silica gel eluting with Ether/CH₂Cl₂ to giveabove product as a white solid. ¹H NMR (CDCl3, 500 MHz): δ 7.0-8.2 (m,10H), 5.72 (dd, 1H, J=3.4 Hz, J=1.1 Hz), 5.44 (t, 1H, J=9.6 Hz), 4.79(d, 1H, J=1.1 Hz), 4.16 (dd, 1H, J=3.4 Hz, J=1.1 Hz), 4.00 (m, 1H), 3.88(m, 1H), 3.82 (m, 1H), 3.66 (m, 2H), 3.31 (m, 2H), 1.82 (m, 2H).

Step C: 2-azidopropoxyl2,4-di-O-benzoyl-3,6-O-(2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl)-β-D-mannopyranoside

The title compound was prepared using procedures analogous to thosedescribed for ML-2 substituting 2.4-benzoyl 3-azidopropoxylβ-D-mannopyranoside for 2-azidoethyl2,4-bis-O-benzoyl-6-O-trityl-α-D-mannopyranoside in Step B. ¹H NMR(CDCl3, 500 MHz): δ 7.0-8.3 (m, 50H), 6.2 (m, 2H), 5.95 (m, 2H), 5.85(m, 2H), 5.70 (m, 1H), 5.35 (m, 2H), 5.22 (s, 1H), 3.0-5.0 (m, 15H),1.90 (m, 2H).

Step D:6-[(2,5-dioxopyrrolidin-1-yl)oxy]-N-(2-{[α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-β-D-mannopyranosyl]oxy}propyl)-6-oxohexanamide

The title compound was prepared using procedures analogous to thosedescribed for ML-2 in Step D-F. UPLC Method B: m/e=787.3816 [M+1];Rt=3.39 min.

Example 80

Synthesis of A1 protected insulin is described.

In an appropriate sized container, insulin is suspended at rt in anorganic solvent, e.g., DMSO, in the presence of a base, e.g., TEA. Themixture is allowed to stir gently until insulin completely dissolved. Tothe resulting solution was added protecting reagent, e.g., ethyltrifluoroacetate or 9-fluorenylmethyl pentafluorophenyl carbonate, neator in solution of organic solvents, such as DMSO or DMF. After UPLCchromatogram shows that a substantial portion of the reaction mixturehas converted into A1-protected insulin, the reaction mixture may besubjected directly for reverse phase HPLC purification (Waters C4 250×50mm column, 10 μm, 1000 Å column or Kromasil C8 250×50 mm, 10 μm, 100 Åcolumn; Buffer A: 0.05-0.1% TFA in deionized water; Buffer B: 0.05-0.1%TFA in AcCN), or the reaction may be quenched by careful dilution withcold acidic H₂O (20×, pH about 3.0) at 0° C. and its pH is adjusted to afinal pH of 2.5 using 1 N HCl (and 0.1 N NaOH if needed). The solutionmay first be concentrated by ultrafiltration, either through atangential flow filtration (TFF) system or using Amicorn Ultra-15Centrifugal Units, with 1K, 3K or 10K MWCO membrane. The concentratedsolution is then subjected to reverse phase HPLC purification (Waters C4250×50 mm column, 10 μm, 1000 Å column or Kromasil C8 250×50 mm, 10 μm,100 Å column; Buffer A: 0.05-0.1% TFA in deionized water; Buffer B:0.05-0.1% TFA in AcCN). Fractions containing the title conjugate arecombined and freeze-dried or buffer exchanged using TFF system and/orAmicorn Ultra-15 to give the N^(A1) protected insulin.

Example 81

The synthesis of N^(A1)-Trifluoroacetyl insulin is described.

In a 100 round bottom flask is charged with insulin (300 mg, 0.052mmol), to which was added 8 mL DMSO, then TEA (43.4 mg, 0.429 mmol). Themixture is gently stirred at rt for about 30 minute until a clearsolution is obtained. To the resulting solution is added ethyltrifluroacetate (35.2 mg, 0.248 mml) After stirring at rt for 4 hr, themixture is diluted carefully with H₂O (100 mL, pH=3.00). After itsvolume is reduced to 20 mL using 10 MWCO Amicon Ultra-15 Centrifugaltubes, the resulting solution is purified by HPLC (Kromasil® C8 10 μm,100 Å, 50×250 mm column at 210 nm, flow rate at 85 mL/min, 0.05% TFA inAcCN/H₂O, 26% AcCN to 37% AcCN in H₂O, 20 min ramp). Desired fractionswere combined and freeze-dried to give the N^(A1)-Trifluoroacetylinsulin. UPLC Method A: m/e=1476.55 [(M+4)/4]; Rt=3.62 min.

Example 82

This example shows the synthesis of IOC-143.

To a solution of N^(A1)-Trifluoroacetyl Human Insulin (77.7 mg, 0.013mmol) in DMSO (1.2 mL) at rt was added TEA (18μL, 0.132 mmol) and asolution of ML-11 (30.2 mg, 0.039 mmol) in DMSO (300μL). After stirringat rt for 4 hours, the mixture was added to AcCN (40 mL). Precipitatewas collected through centrifugation. The collected solid was dissolvedin water (5 mL, pH=3.00) and the mixture was cooled down to 0° C., towhich a solution of NH₄OH (5 mL, 28% in water) was added. The mixturewas stirred at 0° C. for 2 hr and then diluted with water (20 mL,pH=3.00). The volume of the resulting solution was reduced to 5 mL using10K MWCO Amicon Ultra-15 Centrifugal Filter Units, and was furtherdiafiltrated with water (100 mL, pH=3.00) to final volume about 7.5 mL,which was purified by HPLC to give the IOC-143. UPLC Method A: Rt=3.58min; m/e=1801.906.

Example 83

The synthesis of N^(A1)-[(9H-Fluoren-9-ylmethoxy)carbonyl]Human Insulin(N¹-Fmoc Insulin) is described.

Insulin (1.5 g, 0.258 mmol) was dissolved in DMSO (6 mL) in a 20 mlscintillation vial. To the insulin solution was added 9-fluorenylmethylpentafluorophenyl carbonate (0.105 g, 0.258 mmol) in DMSO (1 mL). Themixture was stirred for 15 minutes.

Product was purified by Gilson HPLC chromatography on a C-4 ReversePhase column. The desired fractions (the first-eluting monomer) werecollected and lyophilized to give the desired Nα^(A1)-Fmoc Insulinproduct. UPLC MS (C4, 5 minutes): 1508.37 (M+4/4) at 4.47 minutes.

Example 84

Synthesis of A1, B29 protected insulin or A1,B28 protected insulinlispro is described.

In an appropriate sized container, insulin is suspended at rt in anorganic solvent or mixed aq/organic solvents, e.g., DMSO, in thepresence of a base, e.g., TEA. The mixture is allowed to stir gentlyuntil insulin completely dissolved. To the resulting solution is addedprotecting reagent, e.g., ethyl trifluoroacetate or 9-fluorenylmethylpentafluorophenyl carbonate, neat or in solution of organic solvents,such as DMSO or DMF. After UPLC chromatogram shows that a substantialportion of the reaction mixture has converted into A1,B29-protectedinsulin (A1,B28-protected insulin lispro). The reaction mixture may besubjected directly to reverse phase HPLC purification (Waters C4 250×50mm column, 10 μm, 1000 Å column or Kromasil C8 250×50 mm, 10 μm, 100 Åcolumn; Buffer A: 0.05-0.1% TFA in deionized water; Buffer B: 0.05-0.1%TFA in AcCN), or the reaction may be quenched by careful dilution withcold acidic H₂O (20×, pH ˜3.0) at 0° C. and its pH is adjusted to afinal pH of 2.5 using 1 N HCl (and 0.1 N NaOH if needed). The solutionmay first be concentrated by ultrafiltration, either through atangential flow filtration (TFF) system or using Amicon Ultra-15Centrifugal Units, with 1K, 3K or 10K MWCO membrane. The concentratedsolution is then subjected to reverse phase HPLC purification (Waters C4250×50 mm column, 10 μm, 1000 Å column or Kromasil C8 250×50 mm, 10 μm,100 Å column; Buffer A: 0.05-0.1% TFA in deionized water; Buffer B:0.05-0.1% TFA in AcCN). Fractions containing the title conjugate arecombined and freeze-dried or buffer exchanged using TFF system and/orAmicon Ultra-15 to give the title product.

Example 85

Synthesis of N^(A1), N^(εB29)-Bis(trifluoroacetyl)Human Insulin isdescribed.

In a 100 round bottom flask was charged with human insulin (300 mg,0.052 mmol), to which was added AcCN (6.0 mL), water (6.0 mL), and DIPEA(1.5 mL, 8.59 mmol). To the resulting mixture at 0° C. was added ethyltrifluroacetate (0.9 mL, 7.54 mml). After stirring at 0° C. for 2 hr,the mixture was purified by HPLC (Kromasil® C8 10 μm, 100 Å, 50×250 mmcolumn at 210 nm, flow rate at 85 mL/min, 0.05% TFA in AcCN/H₂O, 27%AcCN to 37% AcCN in H₂O, 20 min ramp). Desired fractions were combinedand freeze-dried to give the N^(A1), N^(εB29)-Bis(trifluoroacetyl)HumanInsulin. UPLC Method A: m/e=1500.677 [(M+4)/4]; Rt=3.87 min.

Example 86

Synthesis of N^(A1), N^(εB29)-Bis[(9H-Fluoren-9-ylmethoxy)carbonyl]HumanInsulin is described.

In a 20 mL scintillation vial, human insulin (1.19 g, 0.205 mmol) andTEA (257 μL, 1.844 mmol) was dissolved in DMSO (10 mL). To this insulinsolution was added1-{[(9H-fluoren-9-ylmethoxy)carbonyl]oxy}pyrrolidine-2,5-dione (207 mg,0.615 mmol) in DMSO (2 mL). After stirring at rt for 30 min, thereaction was quenched by the addition of HCl (1.84 mL, 1.844 mmol, 1.0M). The resulting mixture was purified by reverse phase HPLCchromatography. The desired fractions were collected and lyophilized togive the N^(A1), N^(εB29)-Bis[(9H-Fluoren-9-ylmethoxy)carbonyl]HumanInsulin. UPLC Method A: m/e=1564.04 [(M+4/4)]; Rt=4.41 min.

Example 87 Synthesis of Conjugates with Same Linker-Oligosaccharides onN^(A1) and N^(εB29) of Insulin

In an appropriate sized container, insulin is suspended at rt in anorganic solvent, e.g., DMSO, in the presence of a base, e.g., TEA. Themixture is allowed to stir gently until insulin completely dissolved. Ina separate vial, an activated ester intermediate is dissolved in anorganic solvent, e.g., DMSO, at rt. Aliquots of the solution of theactivated ester is added over a period of time to the solutioncontaining insulin until UPLC chromatogram shows that all of theunmodified insulin has reacted and that a substantial portion of thereaction mixture has converted into A1,B29-conjugated insulin. Thereaction is quenched by the addition of an amine nucleophile, e.g.,2-aminoethanol. The reaction solution is stirred at rt for 30 min. Theresulting solution is carefully diluted with cold H₂O (20×) at 0° C. andits pH is adjusted to a final pH of 2.5 using 1 N HCl (and 0.1 N NaOH ifneeded). The solution is first concentrated by ultrafiltration, eitherthrough a tangential flow filtration (TFF) system or using AmiconUltra-15 Centrifugal Units, with 1K, 3K or 10K MWCO membrane. Theconcentrated solution is usually first subjected to ion exchangechromatography (PolySULFOETHYL A column, PolyLC Inc., 250×21 mm, 5 m,1000 Å; Buffer A: 0.1%(v/v)H₃PO₄/25% AcCN; Buffer B: 0.1%(v/v)H₃PO₄/25%AcCN/0.5 M NaCl). Fractions containing A1,B29-conjugate with desiredpurity are combined and concentrated using TFF system or AmiconUltra-15. The resulting solution is then further purified by reversephase HPLC (Waters C4 250×50 mm column, 10 μm, 1000 Å column or KromasilC8 250×50 mm, 10 μm, 100 Å column; Buffer A: 0.05-0.1% TFA in deionizedwater; Buffer B: 0.05-0.1% TFA in AcCN). Fractions containing the titleconjugate are combined and freeze-dried or buffer exchanged using TFFsystem and/or Amicon Ultra-15 to give the title product.

Example 88 Synthesis of Conjugates with Linker-Oligosaccharide on N^(A1)of Insulin

In an appropriate sized container, insulin is suspended at rt in anorganic solvent, e.g., DMSO, in the presence of a base, e.g., TEA. Themixture is allowed to stir gently until insulin completely dissolved. Ina separate vial, an activated ester intermediate is dissolved in anorganic solvent, e.g., DMSO, at rt. Aliquots of the solution of theactivated ester is added over a period of time to the solutioncontaining insulin until UPLC chromatogram shows that most of theunmodified insulin has reacted and that a substantial portion of thereaction mixture has converted into A1-conjugated insulin. The reactionis quenched by the addition of an amine nucleophile, e.g.,2-aminoethanol. The reaction solution is stirred at rt for 30 min. Theresulting solution is carefully diluted with cold H₂O (20×) at 0° C. andits pH is adjusted to a final pH of 2.5 using 1 N HCl (and 0.1 N NaOH ifneeded). The solution is first concentrated by ultrafiltration, eitherthrough a tangential flow filtration (TFF) system or using AmiconUltra-15 Centrifugal Units, with 1K, 3K or 10K MWCO membrane. Theconcentrated solution is usually first subjected to ion exchangechromatography (PolySULFOETHYL A column, PolyLC Inc., 250×21 mm, 5 μm,1000 Å; Buffer A: 0.1%(v/v)H₃PO₄/25% AcCN; Buffer B: 0.1%(v/v)H₃PO₄/25%AcCN/0.5 M NaCl). Fractions containing A1-conjugate with desired purityare combined and concentrated using TFF system or Amicon Ultra-15. Theresulting solution is then further purified by reverse phase HPLC(Waters C4 250×50 mm column, 10 μm, 1000 Å column or Kromasil C8 250×50mm, 10 μm, 100 Å column; Buffer A: 0.05-0.1% TFA in deionized water;Buffer B: 0.05-0.1% TFA in AcCN). Fractions containing the titleconjugate are combined and freeze-dried or buffer exchanged using TFFsystem and/or Amicon Ultra-15 to give the title product.

Example 89 Synthesis of Conjugates with Linker-Oligosaccharide on N^(B1)of Insulin

N^(B1) insulin conjugate may be prepared according to Example 50. Or itmay be prepared using protected insulin as substrate:

In an appropriate sized container, protected insulin, e.g.,N^(A1),N^(εB29)-bis[(9H-fluoren-9-ylmethoxy)carbonyl]- or N^(A1),N^(εB29)-bis(trifluoroacetyl)human insulin is suspended at rt in anorganic solvent, e.g., DMSO, in the presence of a base, e.g., TEA. Themixture is allowed to stir gently until protected insulin completelydissolved. In a separate vial, an activated ester intermediate isdissolved in an organic solvent, e.g., DMSO, at rt. Aliquots of thesolution of the activated ester is added over a period of time to thesolution containing insulin until UPLC chromatogram shows that all ofthe unmodified insulin has reacted and that a substantial portion of thereaction mixture has converted into B1-conjugated protected insulin. Thereaction is quenched at low temperature by the addition of excess amountof an amine nucleophile, e.g., 2-aminoethanol or ammonia. The reactionsolution is stirred at low temperature until UPLC chromatogram indicatedcomplete removal of the protecting group. The resulting solution iscarefully diluted with cold H₂O (20×) at 0° C. and its pH is adjusted toa final pH of 2.5 using 1 N HCl (and 0.1 N NaOH if needed). The solutionis first concentrated by ultrafiltration, either through a tangentialflow filtration (TFF) system or using Amicon Ultra-15 Centrifugal Units,with 1K, 3K or 10K MWCO membrane. The concentrated solution is usuallyfirst subjected to ion exchange chromatography (PolySULFOETHYL A column,PolyLC Inc., 250×21 mm, 5 μm, 1000 Å; Buffer A: 0.1%(v/v)H₃PO₄/25% AcCN;Buffer B: 0.1%(v/v)H₃PO₄/25% AcCN/0.5 M NaCl). Fractions containingB1-conjugate with desired purity are combined and concentrated using TFFsystem or Amicon Ultra-15. The resulting solution is then furtherpurified by reverse phase HPLC (Waters C4 250×50 mm column, 10 μm, 1000Å column or Kromasil C8 250×50 mm, 10 μm, 100 Å column; Buffer A:0.05-0.1% TFA in deionized water; Buffer B: 0.05-0.1% TFA in AcCN).Fractions containing the title conjugate are combined and freeze-driedor buffer exchanged using TFF system and/or Amicon Ultra-15 to give thetitle product.

Example 90 Synthesis of Conjugates with Linker-Oligosaccharide onN^(εB29) of Insulin

In an appropriate sized container, insulin is dissolved, with gentlestirring, at rt in a mixed solvent: 2:3 v/v 0.1 MNa₂CO³:AcCN. After themixture cleared, the pH is adjusted to the value of 10.5-10.8 usingalkaline solution, e.g., 0.1 NNaOH. In a separate vial, an activatedester intermediate is dissolved in an organic solvent, e.g., DMSO, atrt. Aliquots of the solution of the activated ester is added over aperiod of time to the solution containing insulin until UPLCchromatogram shows that most of the unmodified insulin has reacted andthat a substantial portion of the reaction mixture has converted intoB29-conjugated insulin. The reaction is quenched by the addition of anamine nucleophile, e.g., 2-aminoethanol. The reaction solution isstirred at rt for 30 min. The resulting solution is carefully dilutedwith cold H₂O (20×) at 0° C. and its pH is adjusted to a final pH of 2.5using 1 N HCl (and 0.1 N NaOH if needed). The solution is firstconcentrated by ultrafiltration, either through a tangential flowfiltration (TFF) system or using Amicon Ultra-15 Centrifugal Units, with1K, 3K or 10K MWCO membrane. The concentrated solution is usually firstsubjected to ion exchange chromatography (PolySULFOETHYL A column,PolyLC Inc., 250×21 mm, 5 μm, 1000 Å; Buffer A: 0.1%(v/v)H₃PO₄/25% AcCN;Buffer B: 0.1%(v/v)H₃PO₄/25% AcCN/0.5 M NaCl). Fractions containingB29-conjugate with desired purity are combined and concentrated usingTFF system or Amicon Ultra-15. The resulting solution is then furtherpurified by reverse phase HPLC (Waters C4 250×50 mm column, 10 μm, 1000Å column or Kromasil C8 250×50 mm, 10 μm, 100 Å column; Buffer A:0.05-0.1% TFA in water; Buffer B: 0.05-0.1% TFA in AcCN). Fractionscontaining the title conjugate are combined and freeze-dried or bufferexchanged using TFF system and/or Amicon Ultra-15 to give the titleproduct.

Example 91 Synthesis of Conjugates with Same Linker-Oligosaccharides onN^(B1) and N^(εB29) of Insulin

In an appropriate sized container, protected insulin, e.g.,N^(A1)-(9H-fluoren-9-ylmethoxy)carbonyl- orN^(A1)-(trifluoroacetyl)human insulin is suspended at rt in an organicsolvent, e.g., DMSO, in the presence of a base, e.g., TEA. The mixtureis allowed to stir gently until protected insulin completely dissolved.In a separate vial, an activated ester intermediate is dissolved in anorganic solvent, e.g., DMSO, at rt. Aliquots of the solution of theactivated ester is added over a period of time to the solutioncontaining insulin until UPLC chromatogram shows that all of theunmodified protected insulin has reacted and that a substantial portionof the reaction mixture has converted into B1,B29-conjugated protectedinsulin. The reaction is quenched at low temperature by the addition ofexcess amount of an amine nucleophile, e.g., 2-aminoethanol or ammonia.The reaction solution is stirred at low temperature until UPLCchromatogram indicated complete removal of the protecting group. Theresulting solution is carefully diluted with cold H₂O (20×) at 0° C. andits pH is adjusted to a final pH of 2.5 using 1 N HCl (and 0.1 N NaOH ifneeded). The solution is first concentrated by ultrafiltration, eitherthrough a tangential flow filtration (TFF) system or using AmiconUltra-15 Centrifugal Units, with 1K, 3K or 10K MWCO membrane. Theconcentrated solution is usually first subjected to ion exchangechromatography (PolySULFOETHYL A column, PolyLC Inc., 250×21 mm, 5 μm,1000 Å; Buffer A: 0.1%(v/v)H₃PO₄/25% AcCN; Buffer B: 0.1%(v/v)H₃PO₄/25%AcCN/0.5 M NaCl). Fractions containing B1,B29-conjugate with desiredpurity are combined and concentrated using TFF system or AmiconUltra-15. The resulting solution is then further purified by reversephase HPLC (Waters C4 250×50 mm column, 10 μm, 1000 Å column or KromasilC8 250×50 mm, 10 μm, 100 Å column; Buffer A: 0.05-0.1% TFA in deionizedwater; Buffer B: 0.05-0.1% TFA in AcCN). Fractions containing the titleconjugate are combined and freeze-dried or buffer exchanged using TFFsystem and/or Amicon Ultra-15 to give the title product.

Example 92 Synthesis of Conjugates with Same Linker-Oligosaccharides onN^(A1), N^(B1), and N^(εB29) of Insulin

In an appropriate sized container, insulin is suspended at rt in anorganic solvent, e.g., DMSO, in the presence of a base, e.g., TEA. Themixture is allowed to stir gently until insulin completely dissolved. Ina separate vial, an activated ester intermediate is dissolved in anorganic solvent, e.g., DMSO, at rt. Aliquots of the solution of theactivated ester is added over a period of time to the solutioncontaining insulin until UPLC chromatogram shows that all of theunmodified insulin has reacted and that a substantial portion of thereaction mixture has converted into A1-, B1-, and B29-conjugatedinsulin. The reaction is quenched by the addition of an aminenucleophile, e.g., 2-aminoethanol. The reaction solution is stirred atrt for 30 min. The resulting solution is carefully diluted with cold H₂O(20×) at 0° C. and its pH is adjusted to a final pH of 2.5 using 1 N HCl(and 0.1 N NaOH if needed). The solution is first concentrated byultrafiltration, either through a tangential flow filtration (TFF)system or using Amicon Ultra-15 Centrifugal Units, with 1K, 3K or 10KMWCO membrane. The concentrated solution is usually first subjected toion exchange chromatography (PolySULFOETHYL A column, PolyLC Inc.,250×21 mm, 5 μm, 1000 Å; Buffer A: 0.1%(v/v)H₃PO₄/25% AcCN; Buffer B:0.1%(v/v)H₃PO₄/25% AcCN/0.5 M NaCl).

Fractions containing A1, B1, B29-conjugate with desired purity arecombined and concentrated using TFF system or Amicon Ultra-15. Theresulting solution is then further purified by reverse phase HPLC(Waters C4 250×50 mm column, 10 μm, 1000 Å column or Kromasil C8 250×50mm, 10 μm, 100 Å column; Buffer A: 0.05-0.1% TFA in deionized water;Buffer B: 0.05-0.1% TFA in AcCN). Fractions containing the titleconjugate are combined and freeze-dried or buffer exchanged using TFFsystem and/or Amicon Ultra-15 to give the title product.

Example 93 Synthesis of Conjugates with DifferentLinker-Oligosaccharides on N^(A1) and N^(εB29) of Insulin

In an appropriate sized container, N^(εB29)-conjugated insulin issuspended at rt in an organic solvent, e.g., DMSO, in the presence of abase, e.g., TEA. The mixture is allowed to stir gently until insulincompletely dissolved. In a separate vial, an activated esterintermediate was dissolved in an organic solvent, e.g., DMSO, at rt.Aliquots of the solution of the activated ester are added over a periodof time to the solution containing insulin until UPLC chromatogram showsthat all of the unmodified insulin had been reacted and that asubstantial portion of the reaction mixture had been converted intoA1,B29-conjugated insulin. The reaction is quenched by the addition ofan amine nucleophile, e.g., 2-aminoethanol. The reaction solution isstirred at rt for 30 min. The resulting solution is carefully dilutedwith cold H₂O (20×) at 0° C. and its pH was adjusted to a final pH of2.5 using 1 N HCl (and 0.1 N NaOH if needed). The solution is firstconcentrated by ultrafiltration, either through a tangential flowfiltration (TFF) system or using Amicon Ultra-15 Centrifugal Units, with1K, 3K or 10K MWCO membrane. The concentrated solution may be firstsubjected to ion exchange chromatography (PolySULFOETHYL A column,PolyLC Inc., 250×21 mm, 5 μm, 1000 Å; Buffer A: 0.1%(v/v)H₃PO₄/25% AcCN;Buffer B: 0.1%(v/v)H₃PO₄/25% AcCN/0.5 M NaCl). Fractions containingA1,B29-conjugate with desired purity are combined and concentrated usingTFF system or Amicon Ultra-15. The resulting solution is then furtherpurified by reverse phase HPLC (Waters C4 250×50 mm column, 10 μm, 1000Å column or Kromasil C8 250×50 mm, 10 μm, 100 Å column; Buffer A:0.05-0.1% TFA in deionized water; Buffer B: 0.05-0.1% TFA in AcCN).Fractions containing the title conjugate are combined and freeze-driedor buffer exchanged using TFF system and/or Amicon Ultra-15 to give thetitle product.

Example 94 Synthesis of Conjugates with Same Linker-Oligosaccharides onN^(B1) and N^(εB29) of Insulin

In an appropriate sized container, protected insulin, e.g.,N^(εB29)-(9H-fluoren-9-ylmethoxy)carbonyl- orN^(A1)-(trifluoroacetyl)human insulin is suspended at rt in an organicsolvent, e.g., DMSO, in the presence of a base, e.g., TEA. The mixtureis allowed to stir gently until protected insulin completely dissolved.In a separate vial, an activated ester intermediate is dissolved in anorganic solvent, e.g., DMSO, at rt. Aliquots of the solution of theactivated ester is added over a period of time to the solutioncontaining insulin until UPLC chromatogram shows that all of theunmodified protected insulin has reacted and that a substantial portionof the reaction mixture has converted into A1,B1-conjugated protectedinsulin. The reaction is quenched at low temperature by the addition ofexcess amount of an amine nucleophile, e.g., 2-aminoethanol or ammonia.The reaction solution is stirred at low temperature until UPLCchromatogram indicated complete removal of the protecting group. Theresulting solution is carefully diluted with cold H₂O (20×) at 0° C. andits pH is adjusted to a final pH of 2.5 using 1 N HCl (and 0.1 N NaOH ifneeded). The solution is first concentrated by ultrafiltration, eitherthrough a tangential flow filtration (TFF) system or using AmiconUltra-15 Centrifugal Units, with 1K, 3K or 10K MWCO membrane. Theconcentrated solution is usually first subjected to ion exchangechromatography (PolySULFOETHYL A column, PolyLC Inc., 250×21 mm, 5 μm,1000 Å; Buffer A: 0.1%(v/v)H₃PO₄/25% AcCN; Buffer B: 0.1%(v/v)H₃PO₄/25%AcCN/0.5 M NaCl). Fractions containing B1,B29-conjugate with desiredpurity are combined and concentrated using TFF system or AmiconUltra-15. The resulting solution is then further purified by reversephase HPLC (Waters C4 250×50 mm column, 10 μm, 1000 Å column or KromasilC8 250×50 mm, 10 μm, 100 Å column; Buffer A: 0.05-0.1% TFA in deionizedwater; Buffer B: 0.05-0.1% TFA in AcCN). Fractions containing the titleconjugate are combined and freeze-dried or buffer exchanged using TFFsystem and/or Amicon Ultra-15 to give the title product.

Example 95

Synthesis of N^(εB29)-(trifluoroacetyl)Human Insulin is described

In a 100 round bottom flask was charged with human insulin (200 mg,0.034 mmol), to which was added AcCN (4.0 mL), water (4.0 mL), and TEA(0.5 mL, 3.44 mmol). To the resulting mixture at 0° C. was added ethyltrifluroacetate (0.41 mL, 3.44 mml) After stirring at 0° C. for 30 min,the mixture was diluted with water (20 mL, pH ˜3.0). After acidify theresulting solution until pH ˜2.5 carefully, the mixture was purified byHPLC (Delta Pak C4 15 μm, 300 Å, 50×250 mm column at 210 nm, flow rateat 85 mL/min, 0.05% TFA in AcCN/H2O, 27% AcCN to 37% AcCN in H2O, 20 minramp). Desired fractions were combined and freeze-dried to give thetitle compound. UPLC Method A: m/e=1476.5012 [(M+4)/4]; Rt=3.71 min.

Example 96 Synthesis of IOC-3, Human Insulin Conjugated at B1 and B29 toLinker ML-7

N^(αA1)-Tfa-Insulin (60 mg, 0.01 mmol) was dissolved in 1 ml DMSO at rt,to this solution was added triethylamine (10.3 mg, 0.102 mmol), ML-7(18.2 mg, 0.023 mmol) was dissolved in 100 uL DMSO and added to thereaction mixture. After stirring at rt for 4 hours, the mixture wasadded to 40 ml AcCN. Precipitate was formed and collected bycentrifugation. The collected solid was dissolved in 5 mL PH=3.00 DIwater and cooled down to 0° C., then 5 mL NH₄OH (28% in water) was addedto the water solution, the mixture was stirred at 0° C. for 2 hours andthen diluted with 20 mL DI water PH=3.00. The mixtures was concentrateddown to 5 mL with a 10K membrane Amicon centrifuge tube, and was furtherdiafiltrated with 100 mL PH=3.00 DI water to a final volume about 7.5 mLand purified by prep HPLC. HPLC conditions were as follow: Kromasil® C810 μm, 100 Å, 50×250 mm column at 210 nm, flow rate at 85 mL/min, 0.05%TFA in AcCN/H₂O, 26% AcCN to 32% AcCN in H2O, 25 min ramp, collected thefractions and lyophilized to powder. (38.8 mg, yield 52.4%)1784.76[M+4]/4, t_(R)=3.435

Example 97

This example shows the preparation of an insulin oligosaccharideconjugate (IOC-123) in which oligosaccharide linker ML-11 is linked tothe NH₂ group at positions B1 and B29 of human insulin.

N^(αA1)-Fmoc insulin (80 mg, 0.014 mmol) and linker ML-11 (100 mg, 0.068mmol) were warmed up to room temperature for 30 minutes. To theN^(αA1)-Fmoc insulin (80 mg, 0.014 mmol) in DMSO (1.00 mL) in a 20 mLvial was added triethylamine (18.95 μL, 0.136 mmol). ML-11 (100 mg,0.068 mmol) in DMSO (0.90 mL) was added in to the reaction vial in threeequal portions and 50 minutes interval. The reaction was quenched byadding 2-aminoethanol (103 μL, 1.700 mmol) and stir the mixture at roomtemperature for 20 min. The mixture was diluted into H₂O (10 mL) at 0°C. The pH of the reaction mixture is adjusted to be about 2.5 using 1 NHCl.

The crude product was first purified by ion exchange Chromatography. Thedesired fractions were concentrated using Amicon Ultra CentrifugelFilters or lyophilized overnight and then further purified by reversephase prepare HPLC (Gilson C-4 column) The combined desired fractionswere lyophilized to produce a solid. Then the solid was dissolved inwater and the pH adjusted to 7 using 0.1N NaOH solution to provide asolution of IOC-123.

Example 98

This example illustrates the synthesis of IOC-113 (N^(A1),N^(βB29)-Bis{6-[cis-3,5-bis({2-[(α-L-fucopyranosyl)oxy]ethyl}carbamoyl)piperidin-1-yl]-6-oxohexanoyl}human insulin) in which the A1 and B1 positions of human insulin areconjugated to ML-17.

To a 20 mL scintillation vial containing human insulin (105 mg, 0.018mmol) at room temperature was added DMSO (1 mL) and DIPEA (35.1 mg,0.271 mmol). The mixture was allowed to stir gently until insulindissolved. In a separate vial, linker ML-17 (35.1 mg, 0.045 mmol) wasdissolved in DMSO (0.9 mL) at room temperature. To the solutioncontaining human insulin was added the solution of ML-17 in three equalportions in 50 minute intervals. The reaction was quenched by adding2-aminoethanol (34 μL, 0.42 mmol). After stirring at room temperaturefor 20 minutes, the resulting mixture was carefully diluted with coldH₂O (4 mL) at 0° C. The pH of the resulting mixture was adjusted to afinal pH of 2.5 using 1 N HCl (or 0.1 N NaOH). The mixture was firstsubjected to ion exchange chromatography (PolySULFOETHYL A column,PolyLC Inc., 250×21 mm, 5 μm, 1000 Å; Buffer A: 0.1%(v/v)H₃PO₄/25% AcCN;Buffer B: 0.1%(v/v)H₃PO₄/25% AcCN/0.5 M NaCl). Fractions containing thetitle conjugate as major product were combined and concentrated usingAmicon-15 MWCO 3k or 10k Ultra Centrifugel Filters or freeze-dried afterbeing neutralized to a pH of about 7.0. The resulting solution orreconstituted conjugate solution was then further purified by reversephase prepare HPLC (Waters C4 250×50 mm column, 10 μm, 1000 Å column orKromasil C8 250×50 mm, 10 μm, 100 Å column; Buffer A: 0.05-0.1% TFA indeionized water; Buffer B: 0.05-0.1% TFA in AcCN). Fractionscontaining >95% title conjugate were combined and freeze-dried to givethe title product. UPLCMS (C4, 5 minutes): 1948.66 (M+4/4) at 3.48 min.

Example 99

This example shows the construction of IOC-52(N^(A1)-6-oxo-6-((2-(α-L-fucopyranosyloxy)ethyl)amino)hexanoyl-N^(εB29)-6-((((2-oxo-2-((α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)-2-oxyethyl)amino)(2-oxo-2-((α-L-fucopyranosyloxy)-2-oxoethyl)amino)ethyl)amino)acetamido)-6-oxohexanoylHuman Insulin) in which the A1 and B1 residues of human insulin areconjugated to linkers ML-4 and ML-29, respectively.

Synthesis ofN^(εB29)-6-((((2-oxo-2-((α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)-2-oxyethyl)amino)(2-oxo-2-((α-L-fucopyranosyloxy)-2-oxoethyl)amino)ethyl)amino)acetamido)-6-oxohexanoylHuman Insulin (IOC-58)

Human insulin (1000 mg, 0.172 mmol) was dissolved in aqueous Na₂CO₃ (8.6mL, 0.1 M) and AcCN (5.7 mL). The pH of the resulting solution wasadjusted to 10.5, to which a solution of ML-29 (2,5-Dioxopyrrolidin-1-yl6-((((2-oxo-2-((α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)-2-oxyethyl)amino)(2-oxo-2-((α-L-fucopyranosyloxy)-2-oxoethyl)amino)ethyl)amino)acetamido)-6-oxohexanoate)(289 mg, 0.258 mmol) in DMF (2.9 mL) was added in portion. The reactionprogress was monitored by UPLC-MS and the reaction was quenched byadding ethanolamine (52.1 μL, 0.861 mmol). The reaction mixture wasdiluted with H₂O (15 mL) and pH was adjusted to about 2.5 using 1.0 NHCl solution. The resulting mixture was purified by HPLC (IONChromatography, PolySULFOEthyl A, 9.4×250 mm, gradient 10-45%) (MobilePhase A: 0.1% (v/v) H₃PO₄/25% Acetonitrile in water, Mobile Phase B:0.1% (v/v) H₃PO₄/25% Acetonitrile/0.5M NaCl in water) over 30 minutes,flow rate 15 mL/minutes). The desired fractions were combined,concentrated using 6 Amicon Ultra Centrifuge′ Filters with Utracel 10Kat 3500 RPM at 4° C., and freeze-dried to give the title compound (600mg, 51% yield) as white powder. UPLC Method A: t_(R)=3.58 minutes.[M+4H/4]^(±)=1703.99.

Synthesis ofN^(A1)-6-oxo-6-((2-(α-L-fucopyranosyloxy)ethyl)amino)hexanoyl-N^(εB29)-6-((((2-oxo-2-((α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)-2-oxyethyl)amino)(2-oxo-2-((α-L-fucopyranosyloxy)-2-oxoethyl)amino)ethyl)amino)acetamido)-6-oxohexanoylHuman Insulin

To a solution ofN^(εB29)-6-((((2-oxo-2-((α-D-mannopyranosyl-(1→3)-[α-D-mannopyranosyl-(1→6)]-α-D-mannopyranosyl)-2-oxyethyl)amino)(2-oxo-2-((α-L-fucopyranosyloxy)-2-oxoethyl)amino)ethyl)amino)acetamido)-6-oxohexanoylHuman Insulin (150 mg, 0.022 mmol) and TEA (30.7 μL, 0.22 mmol) in DMSO(1.5 mL) at room temperature was added a solution of2,5-Dioxopyrrolidin-1-yl6-oxo-6-((2-(α-L-fucopyranosyloxy)ethyl)amino)hexanoate (17 mg, 0.040mmol) (ML-4) in DMSO (1.0 mL) in portion. The reaction was quenched byadding ethanolamine (13.32 μL, 0.22 mmol). After stirring at roomtemperature for 15 minutes, the reaction mixture was diluted with H₂O(15 mL) and pH was adjusted to about 2.5 using 1.0 N HCl solution. Theresulting mixture was purified by HPLC (ION Chromatography,PolySULFOEthyl A, 9.4×250 mm, gradient 10-40%) (Mobile Phase A: 0.1%(v/v) H₃PO₄/25% Acetonitrile in water, Mobile Phase B: 0.1% (v/v)H₃PO₄/25% Acetonitrile/0.5MNaCl in water) over 30 minutes, flow rate 15mL/minutes). The desired fractions were combined, concentrated using 2Amicon Ultra Centrifugel Filters with Utracel 10K at 3500 RPM at 4° C.The resulting mixture was purified on HPLC (C4, 50×250 mm, gradient25-30% AcCN in H₂O with 0.1% TFA over 30 minutes, flow rate 85mL/minutes). The desired fractions were combined and freeze-dried togive the title compound (31 mg, 19% yield) as white powder. UPLC MethodA: t_(R)=3.79 min. [M+4H/4]⁺=1783.3.

Example 100 Synthesis of IOC-10, Human Insulin Conjugated at B1 and B29to Linker ML-6

A1 Fmoc insulin (80 mg, 0.014 mmol) and linker ML-6 (100 mg, 0.068 mmol)were warmed up to room temperature for 30 minutes; To A1 Fmoc insulin(80 mg, 0.014 mmol) in DMSO (1.00 mL) in a 20 mL vial was addedtriethylamine (18.95 μL, 0.136 mmol). Linker ML-6 (100 mg, 0.068 mmol)in DMSO (0.90 ml) was added in to the reaction vial in three equalportions and 50 minutes interval. The reaction was quenched by adding2-aminoethanol (103 μl, 1.700 mmol) and stir the mixture at roomtemperature for 20 minutes. The mixture was diluted into H₂O (10 mL) at0° C. The pH of the reaction mixture is adjusted to be about 2.5 using 1N HCl.

The crude product was first purified by ion exchange Chromatography. Thedesired fractions were concentrated using Amicon Ultra CentrifugelFilters or lyophilized overnight and then further purified by reversephase prepare HPLC (Gilson C-4 column) The combined desire fractionswere lyophilized. Then the solid was dissolved in water and the pHadjusted to 7 using 0.1N NaOH solution. The concentration of the samplewas measured by Lambda Bio+UV\Vis Spectrometer at λ 276. UPLCMS (C4, 5minures): 1763.3 (M+4/4) at 3.72 minutes.

Example 101 Synthesis of IOC-226, human insulin conjugated at B1 and B29to linker ML-31 and ML-54, respectively

N^(αA1)-Tfa-Insulin (40 mg, 0.0067 mmol) was dissolved in 1 ml DMSO atrt, to this solution was added DIPEA (0.038 ml, 0.215 mmol), ML-54 (11.5mg, 0.0078 mmol) was dissolved in 115 uL DMSO and added to the reactionmixture. The reaction mixture was stirred at rt for 2 h or until UPLCchromatogram shows a substantial portion of the reaction mixture hadbeen converted into B29-conjugated insulin. Then, second activatedester, ML-31 (16 mg, 0.014 mmol) was dissolved in 160 uL DMSO and addedto the reaction mixture. The reaction mixture was stirred at rt for 16 hor until UPLC chromatogram shows an extensive portion of the reactionmixture had been converted into B1, B29-conjugated insulin. The mixturewas added to 40 ml AcCN. Precipitate was formed and collected bycentrifugation. The collected solid was dissolved in 5 mL PH=3.00 DIwater and cooled down to 0° C., then 5 mL NH₄OH (28% in water) was addedto the water solution, the mixture was stirred at 0° C. for 2 hours andthen diluted with 20 mL DI water PH=3.00. The mixtures was concentrateddown to 3 mL with a 10K membrane Amicon centrifuge tube, and was furtherdiafiltrated with 60 mL PH=3.00 DI water to a final volume about 5 ml.The crude product was first purified by ion exchange Chromatography. Thedesired fractions were concentrated using Amicon Ultra Centrifuge′Filters and then further purified by reverse phase prep HPLC. HPLCconditions were as follow: Kromasil® C8 10 μm, 100 Å, 50×250 mm columnat 210 nm, flow rate at 85 mL/min, 0.05% TFA in AcCN/H₂O, 27% AcCN to33% AcCN in H2O, 25 min ramp, collected the fractions and lyophilized topowder. Then the solid was dissolved in water and the pH adjusted to 7using 0.1N NaOH solution. The concentration of the sample was measuredby Lambda Bio+UV\Vis Spectrometer at λ 276. (16.53 mg, yield 29.2%)1632.54[M+5]/5, t_(R)=3.35

Example 102

Insulin Receptor Binding Assays were performed as follows.

Two competition binding assays were utilized to determine IOC affinityfor the human insulin receptor type B (IR(B)) against the endogenousligand, insulin, labeled with 125_([I]).

Method E: IR binding assay was a whole cell binding method using CHOcells overexpressing human IR(B). The cells were grown in F12 mediacontaining 10% FBS and antibiotics (G418, Penicillin/Strepavidin),plated at 40,000 cells/well in a 96-well tissue culture plate for atleast 8 hrs. The cells were then serum starved by switching to DMEMmedia containing 1% BSA (insulin-free) overnight. The cells were washedtwice with chilled DMEM media containing 1% BSA (insulin-free) followedby the addition of IOC molecules at appropriate concentration in 90 μLof the same media. The cells were incubated on ice for 60 min. The¹²⁵[I]-insulin (10 μL) was added at 0.015 nM final concentration andincubated on ice for 4 hrs. The cells were gently washed three timeswith chilled media and lysed with 30 μL of Cell Signaling lysis buffer(cat #9803) with shaking for 10 min at room temperature. The lysate wasadded to scintillation liquid and counted to determine ¹²⁵[I]-insulinbinding to IR and the titration effects of IOC molecules on thisinteraction.

Method D: IR binding assay was run in a scintillation proximity assay(SPA) in 384-well format using cell membranes prepared from CHO cellsoverexpressing human IR(B) grown in F12 media containing 10% FBS andantibiotics (G418, Penicillin/Strepavidin). Cell membranes were preparedin 50 mM Tris buffer, pH 7.8 containing 5 mM MgCl₂. The assay buffercontained 50 mM Tris buffer, pH 7.5, 150 mM NaCl, 1 mM CaCl₂, 5 mMMgCl₂, 0.1% BSA and protease inhibitors (Complete-Mini-Roche). Cellmembranes were added to WGA PVT PEI SPA beads (5 mg/ml finalconcentration) followed by addition of IOC molecules at appropriateconcentrations. After 5-15 min incubation at room temperature,¹²⁵[I]-insulin was added at 0.015 nM final concentration for a finaltotal volume of 50 μL. The mixture was incubated with shaking at roomtemperature for 1 to 12 hours followed by scintillation counting todetermine ¹²⁵[I]-insulin binding to IR and the titration effects of IOCmolecules on this interaction.

Example 103

Insulin Receptor Phosphorylation Assays were performed as follows.

The insulin receptor phosphorylation assays were performed using thecommercially available Meso Scale Discovery (MSD) pIR assay (See MesoScale Discovery, 9238 Gaithers Road, Gaitherburg, Md.). CHO cells stablyexpressing human IR(B) were in grown in in F12 cell media containing 10%FBS and antibiotics (G418, Penicillin/Strepavidin) for at least 8 hoursand then serum starved by switching to F12 media containing 0.5% BSA(insulin-free) in place of FBS for overnight growth. Cells wereharvested and frozen in aliquots for use in the MSD pIR assay. Briefly,the frozen cells were plated in either 96-well (40,000 cells/well,Methods A and B) or 384-well (10,000 cells/well, Method C) clear tissueculture plates and allowed to recover. IOC molecules at the appropriateconcentrations were added and the cells incubated for 8 min at 37° C.The media was aspirated and chilled MSD cell lysis buffer was added asper MSD kit instructions. The cells were lysed on ice for 40 min and thelysate then mixed for 10 minutes at room temperature. The lysate wastransferred to the MSD kit pIR detection plates. The remainder of theassay was carried out following the MSD kit recommended protocol.

Example 104

Human macrophage mannose receptor 1 (MRC1) Binding Assays were performedas follows.

The competition binding assay for MRC1 utilized a ligand,mannosylated-BSA labeled with the DELFIA Eu-N1-ITC reagent, as reportedin the literature. Anti-MRC1 antibody (25 μl at 2 ng/μl) was added to aProtein G plate that had been washed three times with 100 μl of 50 mMTris buffer, pH 7.5 containing 100 mM NaCl, 5 mM CaCl₂, 1 mM MgCl₂ and0.1% Tween-20 (wash buffer). The antibody was incubated in the plate for1 hr at room temperature with shaking. The plate was washed with washbuffer 3-5 times followed by addition of MRC1 (2 ng/μl finalconcentration) in 25 μl PBS containing 1% stabilizer BSA. The plate wasincubated at room temperature with gentle shaking for 1 hr. The platewas washed three times with wash buffer. The IOC molecules in 12.5 μl ofbuffer at appropriate concentrations were added followed by 12.5 μl ofEu-mannosylated-BSA (0.1 nM final concentration) in 50 mM Tris, pH 7.5containing 100 mM NaCl, 5 mM CaCl₂, 1 mM MgCl₂ and 0.2% stabilizer BSA.The plate was incubated for 2 hrs at room temperature with shakingfollowed by washing three times with wash buffer. Perkin ElmerEu-inducer reagent (25 μl) was added and incubated for 30 min at roomtemperature prior to detection of the Eu signal (Excitation=340 nm:Emission=615 nm). Assay was performed in a 96-well plate with a manualliquid dispense (Method F) or using an automated liquid dispense (MethodG) or in a 384-well plate with an automated dispense (Method H).

Example 105

The following table lists conjugates that were prepared usingappropriate intermediates following one of the General Methods describedabove. These conjugates were characterized using UPLC Method A or UPLCMethod D noted by an asterisk, or UPLC Method G noted by a #, exhibitingeither four charged, i.e. [(M+4)/4], (or five charged, i.e. [(M+5)/5])species of parent compound at certain retention time (Rt). Their invitro biological activities towards insulin receptor (IR) were measuredby either ligand competition assays or functional phosphorylationassays, as described above, labeled as following: Method A: IRphosphorylation assay based on 96-well with manual liquid dispense;Method B: IR phosphorylation assay based on 96-well with automatedliquid dispense; Method C: IR phosphorylation assay based on 384-wellwith automated liquid dispense; Method D: IR binding assay method D;Method E: IR binding assay method E; Method F: MRC1 assay was performedin a 96-well plate with a manual liquid dispense; Method G: MRC1 assaywas performed in a 96-well plate with an automated liquid dispense;Method H: MRC1 assay was performed in a 384-well plate with an automateddispense. The results are shown in Table 1.

TABLE 1 RT Mass [(m + 4)/4 IR Activation IR Binding MRC1 Binding IOC #(min) or (m + 5)/5] IP^(†) (nM) Method IP^(‡) (nM) Method IP^(‡) (nM)Method IOC-1 3.46 1793.16 2.04 B 4.84 D 7.64 H IOC-2 4.49 1951.04 1.83 A4.92 E 3.64 F IOC-3 3.47 1784.12 0.99 A 2.52 D 7.28 F IOC-4 3.50 1784.801.83 C 3.99 D 10.11 G IOC-5 3.37 1618.99 6.80 A 0.89 E 98.17 F IOC-63.37 1618.99 0.27 C 1.82 E 123.70 G IOC-7 3.37 1618.99 1.40 A 0.81 E139.10 F IOC-8 3.11 1793.22 3.71 B 5.16 E 24.17 G IOC-9 3.10 1792.962.17 B 5.19 E 698.00 G IOC-10 3.72 1763.30 0.38 A 0.31 E 9.60 F IOC-113.63 1918.78 12.78 A 3.37 E 5.82 F IOC-12 3.50 1608.55 1.46 A 0.42 E92.46 F IOC-13 3.47 1607.55 4.22 A 1.60 E 106.60 F IOC-14 3.54 1608.083.21 A 0.67 E 8.33 F IOC-15 3.49 1763.12 16.47 A 2.49 E 39.22 F IOC-164.66 1785.46 27.47 B 3.58 E 25.29 G IOC-17 3.31 1770.70 3.91 C 15.33 D25.05 G IOC-18 3.33 1762.05 754.60 C 400.20 D 28.32 G IOC-19 3.261789.60 2.41 C 3.27 D 26.62 G IOC-20 3.30 1774.90 2.51 C 8.05 D 15.83 GIOC-21 3.43 1848.64 2.60 C NA NA 11.65 G IOC-22 3.62 1763.67 18.93 A1.24 E 18.19 F IOC-23 3.49 1757.00 20.34 A 11.67 D 25.05 F IOC-24 3.431940.00 2.02 C 1.33 D 2.47 H IOC-25 3.47 1777.52 2.90 C 3.30 D 10.23 HIOC-26 3.47 1776.66 3.03 C 2.33 D 46.40 H IOC-27 3.71 1918.29 32.43 A7.37 E 1.92 F IOC-28 3.73 1929.29 75.67 A 39.46 E 4.36 F IOC-29 3.761770.22 41.71 A 5.11 E 14.87 F IOC-30 3.70 1940.61 1.83 A 0.59 E 0.44 FIOC-31 3.70 1861.27 13.14 A 1.18 E 4.31 F IOC-32 3.61 1748.08 29.02 A5.11 E 0.47 F IOC-33 3.69 1940.21 24.37 A 4.30 E 0.53 F IOC-34 3.781696.43 0.76 A 0.23 E 32.92 F IOC-35 3.60 1777.42 1.41 C 3.15 D 108.70 GIOC-36 3.56 1926.78 22.33 B 5.66 E 0.54 G IOC-37 3.63 1689.86 3.49 B1.80 E 16.92 G IOC-38 3.63 1689.86 11.79 B 1.43 E 23.23 G IOC-39 3.461909.10 14.24 A 1.61 E 6.90 F IOC-41 3.52 1604.94 6.11 A 0.88 E 113.50 FIOC-42 3.66 1782.89 2.83 B 0.43 E 13.13 G IOC-43 3.53 1955.10 10.98 A2.00 E NA NA IOC-44 3.64 1955.10 56.62 A 2.93 E 0.16 F IOC-45 3.721704.00 4.30 A 0.76 E 11.54 F IOC-46 3.72* 1703.60* 1.45 A 1.50 E 0.18 FIOC-47 3.57 1725.10 16.53 A 1.42 E 20.41 F IOC-48 3.47 1714.00 12.75 A1.88 E 17.79 F IOC-49 3.37 1868.30 15.37 A 1.51 E 1.14 F IOC-50 3.67*1869.90* 37.08 A 6.11 E 0.49 F IOC-51 3.67* 1703.90* 31.45 A 7.79 E 0.37F IOC-52 3.75 1783.30 16.75 A 5.69 D 9.80 F IOC-53 3.41 1697.70 11.31 A1.09 E 45.46 F IOC-54 3.40 1787.30 14.34 A 2.33 E 18.84 F IOC-55 3.401870.50 15.95 A 1.76 E 17.32 F IOC-56 3.68* 1954.90* 34.99 A 4.83 E 1.41F IOC-57 3.63* 1697.30* 49.88 A 8.66 E 1.01 F IOC-58 3.72* 1703.60* 1.44A 0.41 E 7.95 F IOC-59 3.75 1735.50 1.96 A 1.15 E 20.60 F IOC-60 3.291785.21 39.32 A 8.49 D 10.32 F IOC-61 3.65 1615.55 99.94 A 9.48 E NA NAIOC-62 3.51 1637.87 57.38 A 11.53 E NA NA IOC-63 3.70 1750.00 1.90 A0.91 E 29.26 F IOC-64 3.35 1609.35 51.08 A 6.14 E 0.46 F IOC-65 3.421731.60 2.87 A 0.68 E 21.61 F IOC-66 3.38 1926.98 41.29 A 6.88 E 3.62 FIOC-67 3.43 1918.85 34.42 A 6.65 E 44.95 F IOC-68 3.47 1839.78 28.79 A4.33 E 44.61 F IOC-69 3.50 1866.88 62.09 A 14.64 E NA NA IOC-70 3.641632.15 77.13 A 9.62 E NA NA IOC-71 3.74 1746.08 2.41 A 1.37 E 21.47 FIOC-72 3.50 1884.29 43.52 A 26.02 E NA NA IOC-73 3.57 1643.44 70.50 A5.44 E NA NA IOC-74 3.75 1753.44 3.27 A 0.59 E 27.37 F IOC-75 3.651654.71 41.39 A 4.90 E 0.58 F IOC-76 3.76 1760.52 4.28 A 0.49 E 27.14 FIOC-77 3.68 1827.80 57.29 B 6.45 E 20.43 G IOC-78 3.79 1827.70 25.84 B18.09 E 12.74 G IOC-79 3.73 1811.57 13.48 A 1.73 E 49.26 F IOC-80 3.471704.88 5.30 C NA NA 2.83 G IOC-81 3.48 1904.44 4.35 C NA NA 27.13 GIOC-82 4.52 1866.14 2.75 C 2.39 E 16.61 G IOC-83 4.67 1786.54 5.96 C2.62 E 69.77 G IOC-84 3.63 1899.84 22.99 B 3.82 E 4.24 G IOC-85 4.111735.17 4.01 B 0.84 E 31.42 G IOC-86 4.42 1735.24 13.41 B 0.71 E 84.94 GIOC-87 4.34 1859.16 23.47 B 1.43 E 67.94 G IOC-88 3.87 1628.73 15.06 B6.39 E 1.08 G IOC-89 4.10 1871.51 4.15 B 2.28 E 2.06 G IOC-90 3.521892.85 2.92 B 1.13 E 2.63 G IOC-91 3.47 1892.93 32.77 B 8.96 E 4.67 GIOC-92 3.51 1852.31 4.83 B 4.20 E 5.69 G IOC-93 3.80 1871.12 10.75 B1.68 E 4.65 G IOC-94 3.52 1790.30 13.21 B 2.37 E 61.05 G IOC-95 3.781853.48 12.02 B 1.87 E 47.13 G IOC-96 3.49 1690.29 0.76 B 0.66 D 71.49 HIOC-97 3.60 1894.87 25.71 B 2.07 E 56.62 G IOC-98 3.61 1749.43 28.44 B1.11 E 128.10 G IOC-99 3.84 1738.89 16.31 A 1.29 E NA NA IOC-100 3.641603.19 53.03 A 6.55 E NA NA IOC-101 3.62 1728.12 0.75 A 0.34 E 78.00 FIOC-102 3.46 1956.55 0.88 C 0.92 D 1.49 H IOC-103 3.38 1955.25 0.92 C0.68 D 1.31 H IOC-104 3.43 1704.57 0.31 C NA NA 42.85 G IOC-105 3.421703.58 0.41 C NA NA 34.47 G IOC-106 4.56 1706.54 1.22 B 0.79 E 31.90 GIOC-107 4.45 1706.89 21.47 B 0.61 E 75.41 G IOC-108 4.57 1706.90 8.62 B0.83 E 53.19 G IOC-109 3.64 1739.29 136.70 B 9.01 E 0.60 G IOC-110 3.621933.29 242.20 B 13.73 E 2.63 G IOC-111 3.64 1821.80 5.66 A 1.11 E464.30 F IOC-112 3.71 1698.50 6.27 A 1.52 E 335.30 F IOC-113 3.511783.55 2.89 C 1.77 D 20.73 H IOC-114 3.48 1949.31 2.17 C 2.19 D 3.83 HIOC-115 3.62 1693.27 17.80 B 0.68 E 30.92 G IOC-116 3.63 1692.81 2.65 B0.52 E 13.28 G IOC-117 4.28 1693.09 5.47 B 0.46 E 24.12 G IOC-118 4.111933.41 60.22 B 1.77 E 0.63 G IOC-119 4.01 1933.72 4.39 B 0.60 E 0.40 GIOC-120 3.96 1933.70 14.72 B 1.31 E 0.43 G IOC-121 3.58 1772.28 16.43 B1.05 E 1.77 G IOC-122 3.89 1871.38 11.12 A 3.36 E NA NA IOC-129 3.541783.64 0.37 C 0.41 D 22.86 H IOC-130 4.00 1588.44 965.90 B 6.46 E 3.51G IOC-131 4.08 1808.21 23.39 B 3.23 E 15.36 G IOC-132 4.19 1498.92 43.05B 3.55 E 5.09 G IOC-133 4.28 1508.40 66.81 B 3.95 E 5.22 G IOC-134 4.471807.76 5.50 B 1.30 E 11.38 G IOC-135 3.31 1890.75 3.29 C 4.24 D 30.59 HIOC-136 3.28 1688.38 7.83 C 4.25 D 12.71 H IOC-137 3.50 1706.80 0.99 C1.13 D 475.50 H IOC-138 3.34 1833.60 3.35 C 2.25 D 279.60 H IOC-139 3.521706.03 0.30 C 0.30 D 785.30 H IOC-140 3.24 1784.78 2.12 C 3.19 D 235.60H IOC-141 3.48 1949.31 0.82 C 0.91 D 44.34 H IOC-142 3.51 1783.55 0.87 C1.00 D 182.80 H IOC-143 3.58 1777.59 0.51 C 0.38 D 7.17 H IOC-144 3.501763.92 6.63 C 9.24 D 30.46 H IOC-145 3.49 1985.78 6.75 C 15.05 D 2.69 HIOC-146 3.55 1808.57 6.04 C 9.65 D 15.63 H IOC-147 3.53 1971.72 3.12 B20.33 D 5.42 H IOC-148 3.60 1793.57 3.09 B 15.76 D 21.67 H IOC-149 3.471990.59 3.47 B 2.32 D 2.62 H IOC-150 3.54 1811.75 2.82 B 2.10 D 18.08 HIOC-151 3.53 1975.17 1.48 B 6.59 D 3.33 H IOC-152 3.59 1798.10 2.36 B3.12 D 9.50 H IOC-153 3.41 1703.52 0.40 B 0.20 D 1121.00 H IOC-154 3.341792.79 0.76 B 0.52 D 5.32 H IOC-155 3.40 1963.07 2.82 B 4.82 D 3.34 HIOC-156 3.48 1793.12 1.26 B 2.86 D 16.32 H IOC-157 3.41 1964.48 3.22 B2.85 D 4.98 H IOC-158 3.52 1797.41 0.50 B 0.66 D 11.57 H IOC-159 3.501692.99 0.54 B 0.45 D 56.84 H IOC-160 3.54 1678.14 0.59 B 0.80 D 48.55 HIOC-161 3.50 1697.68 0.78 C 0.61 D 39.81 H IOC-162 3.55 1683.65 0.41 B0.18 D 21.28 H IOC-163 3.56 1617.96 0.70 C 1.26 D 95.98 H IOC-164 3.501784.06 1.57 B 7.09 D 24.35 H IOC-165 3.49 1948.95 1.29 B 3.51 D 3.22 HIOC-166 3.53 1935.09 5.36 C 15.14 D 3.85 H IOC-167 3.54 1768.64 1.73 B3.25 D 25.61 H IOC-168 3.43 1778.13 1.20 B 2.76 D 6.28 H IOC-169 3.431940.02 0.93 B 1.94 D 1.40 H IOC-170 3.49 1763.28 1.10 B 2.87 D 7.06 HIOC-171 3.49 1925.61 1.27 B 1.98 D 1.49 H IOC-172 3.43 1782.34 1.49 C7.18 D 8.66 H IOC-173 3.43 1944.07 0.59 B 1.13 D 1.22 H IOC-174 3.481767.66 0.74 B 1.61 D 4.05 H IOC-175 3.43 1930.01 0.89 B 1.95 D 0.58 HIOC-176 3.45 1705.38 0.54 B 0.80 D 21.01 H IOC-177 3.45 1727.38 0.88 B0.84 D 27.85 H IOC-178 3.47 1706.39 0.59 B 1.06 D 17.65 H IOC-179 3.471705.71 0.60 B 0.88 D 28.44 H IOC-180 3.48 1706.43 0.66 B 0.36 D 22.91 HIOC-181 3.53 1578.92 0.37 B 0.12 D 40.56 H IOC-182 3.41 1831.90 1.09 B1.81 D 27.78 H IOC-183 3.54 1579.64 0.65 B 0.63 D 132.80 H IOC-184 3.541579.85 0.59 B 0.12 D 78.63 H IOC-185 3.51 1589.51 0.49 B 0.73 D 61.87 HIOC-186 3.51 1589.98 0.53 C 0.47 D 38.06 H IOC-187 3.53 1579.56 0.48 B0.52 D 42.02 H IOC-188 3.53 1579.74 0.37 B 0.11 D 42.77 H IOC-189 3.441833.23 0.68 B 0.32 D 8.18 H IOC-190 3.54 1579.12 0.20 B 0.17 D 36.60 HIOC-191 3.48 1693.64 0.54 B 0.55 D 32.11 H IOC-192 3.43 1708.06 0.45 B0.27 D 61.62 H IOC-193 3.29 1705.27 2.15 C 4.68 D 1.06 H IOC-194 3.571706.16 2.26 C 4.14 D 4.62 H IOC-195 3.19 1707.01 2.10 C 2.37 D 14.94 HIOC-196 3.15^(#) 1707.84 2.50 C 3.35 D 148.80 H IOC-197 4.09^(#) 1661.241.60 C 1.95 D 15.77 H IOC-198 4.05^(#) 1870.09 12.81 C 20.57 D 1.88 HIOC-199 4.06^(#) 1870.08 9.18 C 15.12 D 4.35 H IOC-200 3.96^(#) 1869.109.67 C 19.76 D 0.48 H IOC-201 4.08^(#) 1664.55 2.23 C 4.64 D 7.98 HIOC-202 4.07^(#) 1875.95 8.67 C 11.91 D 0.58 H IOC-205 3.62^(#) 1776.625.14 C 18.67 D 35.88 H IOC-206 3.49^(#) 1938.42 5.05 C 7.27 D 5.97 HIOC-207 3.56^(#) 1720.43 6.71 C 19.67 D 292.20 H IOC-208 3.56^(#)1854.45 2.08 C 6.94 D 43.47 H IOC-209 3.59^(#) 1586.34 1.25 C 1.14 D709.60 H IOC-210 3.35^(#) 1727.73 6.71 C 15.29 D 1047.00 H IOC-2113.32^(#) 1865.23 8.64 C 34.34 D 645.00 H IOC-212 3.31^(#) 1492.67 31.40A 11.41 D 8.96 F IOC-213 3.33^(#) 1492.86 16.89 C 9.49 D 53.73 H IOC-2143.34^(#) 1783.20 7.12 C 4.11 D 45.82 H IOC-215 3.33^(#) 1782.94 7.94 C4.45 D 65.78 H IOC-216 3.37^(#) 1559.84 24.41 C 24.60 D 25.79 H IOC-2173.35 1559.62 5.17 C 5.52 D 19.99 H IOC-218 3.49 1771.33 0.84 C 1.40 D61.81 H IOC-219 3.73^(#) 1933.61 0.97 C 0.93 D 0.73 H IOC-220 3.41^(#)1783.30 3.83 C 5.60 D 43.99 H IOC-221 3.4^(#) 1787.17 5.78 C 6.62 D52.43 H IOC-222 3.41^(#) 1776.39 4.52 C 5.23 D 40.61 H IOC-223 3.41^(#)1703.87 0.95 C 0.77 D 33.04 H IOC-224 3.43^(#) 1896.33 1.31 C 0.94 D2.28 H IOC-225 3.29^(#) 1632.63 1.29 C 1.42 D 2.45 H IOC-226 3.35^(#)1632.54 1.22 C 0.85 D 2.04 H IOC-227 3.38^(#) 1955.37 1.92 C 0.71 D 0.93H IOC-228 3.43^(#) 1862.43 10.22 C 7.57 D 64.93 H IOC-229 3.42^(#)1870.65 5.09 C 8.08 D 43.66 H IOC-230 3.47 1848.22 9.84 C 7.46 D 46.40 HIOC-231 3.57 1618.65 2.06 C 1.53 D 231.20 H IOC-232 3.48 1783.50 4.12 C4.05 D 21.24 H IOC-233 3.40 1776.58 1.65 C 7.07 D 33.12 H IOC-234 3.511777.03 0.73 C 1.36 D 23.11 H IOC-235 3.43 1783.00 0.60 C 0.56 D 9.01 HIOC-236 3.51 1783.70 3.54 C 8.27 D 27.34 H IOC-237 3.49^(#) 1948.88 2.75C 6.34 D 3.70 H IOC-238 4.02 1807.35 3.54 C 4.02 D 21.88 H IOC-246 3.521970.09 7.74 C 10.05 D 2.08 H IOC-247 3.57 1797.76 2.59 C 9.18 D 21.59 HIOC-248 3.55 1797.82 0.94 C 0.93 D 11.36 H IOC-249 3.51 1938.30 2.96 C6.29 D 2.69 H IOC-250 3.52 1776.47 6.77 C 10.52 D 37.92 H IOC-251 3.581776.50 1.05 C 2.17 D 52.27 H IOC-252 3.47 1776.55 1.61 C 1.21 D 40.68 HIOC-253 3.65 1552.35 0.53 C 0.73 D 1.52 H IOC-255 3.46 1760.10 11.20 C7.24 D 41.82 H IOC-256 3.36 1734.75 3.98 C 3.20 D 52.56 H IOC-257 3.591593.57 0.76 C 0.79 D 440.40 H IOC-258 3.36 1587.43 1.29 C 0.72 D 234.10H IOC-259 3.35 1721.28 3.93 C 4.53 D 76.67 H IOC-260 3.36 1855.61 4.40 C4.97 D 18.08 H IOC-261 3.32 1721.46 4.47 C 2.62 D 280.90 H IOC-262 3.341856.36 4.08 C 3.07 D 102.30 H IOC-263 3.23 1735.74 5.54 C 4.77 D 132.40H IOC-264 3.25 1876.85 6.01 C 9.15 D 34.11 H IOC-265 3.23 1735.38 2.18 C2.32 D 55.74 H IOC-266 3.24 1876.68 2.14 C 4.70 D 9.72 H IOC-267 3.501597.63 1.28 C 1.46 D 217.20 H IOC-268 3.74 1742.53 4.86 C 4.28 D 76.89H IOC-269 3.87 1749.83 4.74 C 3.36 D 167.60 H IOC-270 3.88 1898.28 1.81C 2.84 D 9.74 H IOC-271 4.37 1888.65 1.02 C 9.38 D 7.01 H IOC-272 4.351754.71 0.90 C 5.38 D 17.11 H ^(†)IP is inflection point and correspondsto EC₅₀. ^(‡)IP is inflection point and corresponds to IC₅₀.

Example 106

Effect of Methyl α-D-Mannopyranoside (aMM) on PK and PD of IOCs inNon-Diabetic Minipigs was evaluated.

Male Yucatan miniature pigs, non-diabetic, instrumented with two Jugularvein vascular access ports (VAP), are used in these studies. Animals arefasted overnight prior to the study. On the day of the study, animalsare restrained in slings, and VAPs accessed for infusion and sampling.At t=−60 minutes, a constant infusion of PBS (n=3) or 21.2% α-methylmannose (αMM) (n=3) is started, at a rate of 2.67 mL/kg/hr. Thisinfusion will be maintained for the duration of the study. At t=0 min,and after collecting a baseline blood sample for plasma glucosemeasurement, animals are administered IOC as a single bolus IV. Samplingcontinues for 90 minutes, with final readouts of plasma glucose andcompound levels.

IOCs are formulated at 17-69 nmol/mL in sodium chloride (87 mM), phenol(21 mM), dibasic sodium phosphate (26.5 mM), Osmolality=275 mOsm,pH=7.4; QS with Water for Injection.

Time points for sample collection: −60 min, 0 min, 1 min, 2 min, 4 min,6 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 45 min, 60min, and 90 min.

Blood is collected in K3-EDTA tubes, supplemented with 10 μg/mlAprotinin, and kept on an ice bath until processing, within 30 minutesof collection. After centrifugation at 3000 rpm, 4° C., for 8 min,plasma is collected and aliquoted for glucose measurement using aBeckman Coulter AU480 Chemistry analyzer and for compound levelsmeasurement by LC-MS.

Glucose results are expressed as % changes over baseline values at t=0minutes and are shown for IOC-2, IOC-3, IOC-8, IOC-9, IOC-16, IOC-22,IOC-23, IOC-46, IOC-48, IOC-52, IOC-56, IOC-60, IOC-75, IOC-75, andIOC-76 in FIGS. 1-14, respectively.

PK results for IOC-2, IOC-3, IOC-16, IOC-22, IOC-23, IOC-52, IOC-56, andIOC-60 are shown in FIGS. 15-22, respectively.

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the claims attached herein.

What is claimed:
 1. A method for treating a subject who has diabetescomprising: administering to the subject an amount of a compositioncomprising a conjugate having the formula

wherein insulin refers to human insulin, and a pharmaceuticallyacceptable carrier; wherein said administering treats the diabetes. 2.The method of claim 1, wherein the diabetes is type I diabetes.
 3. Themethod of claim 1, wherein the diabetes is type II diabetes.
 4. Themethod of claim 1, wherein the diabetes is gestational diabetes.